Tracing the phylogenetic history of the Crl regulon through the Bacteria and Archaea genomes

Abstract

Background

Crl, identified for curli production, is a small transcription factor that stimulates the association of the σ^S factor (RpoS) with the RNA polymerase core through direct and specific interactions, increasing the transcription rate of genes during the transition from exponential to stationary phase at low temperatures, using indole as an effector molecule. The lack of a comprehensive collection of information on the Crl regulon makes it difficult to identify a dominant function of Crl and to generate any hypotheses concerning its taxonomical distribution in archaeal and bacterial organisms.

Results

In this work, based on a systematic literature review, we identified the first comprehensive dataset of 86 genes under the control of Crl in the bacterium Escherichia coli K-12; those genes correspond to 40% of the σ^S regulon in this bacterium. Based on an analysis of orthologs in 18 archaeal and 69 bacterial taxonomical divisions and using E. coli K-12 as a framework, we suggest three main events that resulted in this regulon’s actual form: (i) in a first step, rpoS, a gene widely distributed in bacteria and archaea cellular domains, was recruited to regulate genes involved in ancient metabolic processes, such as those associated with glycolysis and the tricarboxylic acid cycle; (ii) in a second step, the regulon recruited those genes involved in metabolic processes, which are mainly taxonomically constrained to Proteobacteria, with some secondary losses, such as those genes involved in responses to stress or starvation and cell adhesion, among others; and (iii) in a posterior step, Crl might have been recruited in Enterobacteriaceae; because its taxonomical pattern constrained to this bacterial order, however further analysis are necessary.

Conclusions

Therefore, we suggest that the regulon Crl is highly flexible for phenotypic adaptation, probably as consequence of the diverse growth environments associated with all organisms in which members of this regulatory network are present.

Electronic supplementary material

The online version of this article (10.1186/s12864-019-5619-z) contains supplementary material, which is available to authorized users.

Background

Gene expression in bacteria is coordinated through the DNA-binding transcription factors (TFs), blocking or allowing the access of the RNA polymerase (RNAP)-sigma factor to the promoter and providing bacteria with the ability to activate or repress multiple genes under different metabolic stimuli or growth conditions. In the bacterium Escherichia coli K-12, seven sigma factors have been experimentally identified, together with around 300 different TFs responsible for recognizing and activating almost all of their genes [1]. Among these, RpoD, or σ⁷⁰, regulates around 40% of the total gene repertoire, whereas alternative sigma factors such as RpoS (σ^S), the master regulator of the stationary-phase response [2], regulate between 5 and 10% of the total genes in E. coli K-12 [3].

Sigma factors and TFs regulate a large diversity of genes, hierarchically organized in regulons [4]. Previous comparative genomics studies have suggested that regulons exhibit considerable plasticity across the evolution of bacterial species [5]. In this regard, comparison of the gene composition of the PhoPQ regulon in E. coli and Salmonella enterica serovar Typhimurium revealed a very small overlap in both species, suggesting a low similarity in composition between the target genes that are regulated by PhoP in S. Typhimurium strains and in E. coli K-12 [6]. Incidentally, this plasticity in bacterial regulons is evidence of lineage-specific modifications [7].

We conducted an exhaustive analysis concerning the conservation of the Crl regulon in Bacteria and Archaea cellular domains, using as a reference the currently known system in E. coli K-12. Contrary to the most common regulatory mechanisms that involve the direct binding to operators or activators, Crl is an RNAP holoenzyme assembly factor that was originally identified in curli production. It is expressed at low temperatures (30 °C) [8] during the transition phase between the exponential and stationary phases, under low osmolarity, as well as in stationary phase [9]. In E. coli, Crl has a global regulatory effect in stationary phase, through σ^S, as it reorganizes the transcriptional machinery [10], stimulating the association of σ^S with the RNAP core, tilting the competition between σ^S and σ⁷⁰ during the stationary phase in response to different stress conditions [11, 12] [8, 13]; its production is concomitant with the accumulation of σ^S [8].

Assembling the different pieces of the Crl regulon and its regulatory network into one global picture is one of our objectives in this work. The evaluation of this regulon in Bacteria and Archaea will provide clues about how the regulation of genes by Crl has been recruited in all the organisms, i.e., if the regulated genes were recruited similar to Crl or if they followed different pathways. To this end, 86 genes under the control of Crl in E. coli K-12 were compiled from exhaustive literature searches. To our knowledge, this is the first attempt to describe the genes regulated by Crl in E. coli K-12; in addition, few Crl homologs were identified among bacterial and archaeal genomes, constrained to Enterobacteriaceae species. Finally, members of the regulon were identified as widely distributed beyond enterobacteria, suggesting that Crl was recruited in a secondary evolutionary event to regulate a specific subset of genes, most likely genes involved in a functional response in enterobacteria to contend against starvation.

Results

86 genes belong to the Crl regulon

Available information regarding the Crl regulon was gathered through an exhaustive review of the literature. In this regard, diverse experimental evidences were considered significant for determining the association between the regulated genes and Crl protein regulator, such as gene expression analysis (transcriptional fusions), mapping of signal intensities (RNA-seq or microarray analysis), and inferences made from a mutant phenotype (mutation of a TF with a visible cell phenotype), among other analyses. Therefore, 86 genes were included in this work as members of the σS sigmulon, of which 37 had already been reported in both RegulonDB and EcoCyc database; whereas, 49 genes identified by microarray data and crl rpoS double mutants [8–16], in previous works were also added (see Additional file 1). From the 86 genes identified as members of this regulon (see Table 1 and Fig. 1), 34 have a σ^S-type promoter experimentally determined and 8 genes have 13 σ^S-type promoters predicted by computational approaches [17]. These 86 genes are organized in 77 transcription units (TUs), where 52% are TUs with only one gene.

Table 1.

Genes regulated by Crl in Escherichia coli

Gene	Bnumber	TU(s)	TFs	Effect of Crl	Evidence	Reference(s)	GO Terms
aat	b0885	aat		+	GEA and IMP	[11]	protein catabolic process, ubiquitin-dependent protein catabolic process via the N-end rule pathway
accB	b3255	accBC	AccB (−), FadR(+)	+	GEA and IMP	[11]	lipid metabolic process, fatty acid metabolic process, fatty acid biosynthetic process
accC	b3256	accBC	AccB (−), FadR(+)	+	GEA and IMP	[11]	lipid metabolic process, fatty acid metabolic process, fatty acid biosynthetic process, metabolic process, negative regulation of fatty acid biosynthetic process, malonyl-CoA biosynthetic process
acnB	b0118	acnB	CRP(+) ArcA(−), Cra(−), Fis (−)	–	IMP	[13]	regulation of translation, propionate catabolic process, 2-methylcitrate cycle, glyoxylate cycle, tricarboxylic acid cycle metabolic process
ada	b2213	ada- alkB	Ada(+/−)	+	GEA	[13]	DNA dealkylation involved in DNA repair, regulation of transcription, cellular response to DNA damage stimulus, metabolic process, methylation, DNA demethylation
allR	b0506	allR		+	MSI	[10]	regulation of transcription, cellular response to DNA damage stimulus, negative regulation of transcription
bfr	b3336	bfd -bfr		+	MSI, IMP	[40]MSI, [11]IMP	iron ion transport, cellular iron ion homeostasis, intracellular sequestering of iron ion, oxidation-reduction process
bglG	b3723	bglG bglG FB	CRP (+), Fis (−), H-NS (−), LeuO (+), RcsB-BglJ (+), StpA (−)	–	MSI, IMP	[16]	regulation of transcription, positive regulation of transcription
bioB	b0775	bioB FCD	BirA (−)	–	IMP	[13]	biotin biosynthetic process
cbpA	b1000	cbpA M	Fis (−)	+	GEA	[13]	protein folding
crl	b0240	crl	Fur (−)	–	MSI, IMP	[12]	regulation of transcription, DNA-templated, cellular protein complex assembly, positive regulation of transcription
csgA	b1042	csgBAC	CpxR (−), CsgD (+), FliZ (−)	+	APPH, MSI, IMP, GEA, IMP	[8] APPH, MSI, IMP, [9] GEA, IMP	cell adhesion, single-species biofilm formation, amyloid fibril formation
csgB	b1041	csgBAC	CpxR(−), CsgD(+), FliZ(−)	+	APPH, MSI, IMP, GEA, IMP	[8] APPH, MSI, IMP, [9] GEA, IMP	cell adhesion, single-species biofilm formation, amyloid fibril formation
csgC	b1043	csgBAC	CpxR (−), CsgD (+), FliZ (−)	+	MSI	[10]
csgD	b1040	csgD EFG	BasR (+), Cra (+), CRP (+), CsgD (+), IHF (+), MlrA (+), OmpR (+), RcdA (+), CpxR(−), FliZ (−), RcsAB (−), RstA (−)	+	IMP	[13]	regulation of single-species biofilm formation
cstA	b0598	cstA	CRP (+)	+	GEA, IMP	[11]	cellular response to starvation
cysP	b2425	cysP UWAM	CysB (+), H-NS (−)	+	MSI	[10]	sulfur compound metabolic process, transport, sulfate transport, sulfate transmembrane transport
djlC (ybeV)	b0649	ybeU -djlC		+	MSI	[10]	positive regulation of ATPase activity
dps	b0812	dps	Fis(−), H-NS(−),IHF(+), MntR(−), OxyR(+)	+	GEA, IMP	[11]	cellular iron ion homeostasis, response to stress, chromosome condensation, response to starvation, oxidation-reduction process
fbaB	b2097	fbaB	Cra(−)	+	GEA, IMP	[11]	glycolytic process, transcription
flgM	b1071	flgMN, flg A M N	CsgD(−)	–	GEA, IMP	[13]	regulation of transcription, bacterial-type flagellum organization, negative regulation of proteolysis, negative regulation of transcription
fliA	b1922	fliA Z -tcyJ	H-NS(+), MatA(−), SutR(−), NsrR(−), CsgD(−), FlhDC(+)	–	IMP	[13]	transcription initiation from bacterial-type RNAP promoter, sporulation resulting in formation of a cellular spore
fur	b0683	fur fldA-uof -fur uof -fur	CRP(+), Fur(−)	+	MSI, IMP	[12]	regulation of transcription, negative regulation of transcription
gadA	b3517	gadA X	AdiY(+), ArcA(+), CRP(−), FNR(−), Fis(−), GadE-RcsB(+), GadW(+−), GadX(+), H-NS(−), RcsB(−), TorR(−)	+	MSI	[10]	glutamate metabolic process, carboxylic acid metabolic process, intracellular pH elevation
gadB	b1493	gadBC	AdiY(+),CRP(−), Fis(−), FliZ(−), GadE(+), GadW(+−), GadX(+), RcsB(+)	+	MSI, GEA	[40]MSI, [13]GEA	glutamate metabolic process, carboxylic acid metabolic process, intracellular pH elevation
gadC	b1492	gadBC	AdiY(+), CRP(−), Fis(−), FliZ(−), GadE(+), GadW(+−), GadX(+), RcsB(+)	+	MSI	[10]	amino acid transmembrane transport, transport, amino acid transport, intracellular pH elevation
gadE	b3512	gadE- mdtEF gadE	ArcA(+), CRP(−), EvgA(+), FliZ(−), GadE(+), GadW(+), GadX(+), H-NS(−), PhoP(+), YdeO(+)	+	MSI	[10]	regulation of transcription
gadW	b3515	gadW	GadW (+), GadX (−), H-NS(−), PhoP (+), SdiA (+), YdeO (+)	+	MSI	[10]	regulation of transcription, cellular response to DNA damage stimulus
glgS	b3049	glgS	CRP(+)	+	GEA	[13]	glycogen biosynthetic process, positive regulation of cellular carbohydrate metabolic process, negative regulation of single-species biofilm formation on inanimate substrate, negative regulation of bacterial-type flagellum-dependent cell motility
glnH	b0811	glnH PQ	IHF(+), NtrC(+/−)	+	GEA, IMP	[11]	transport, amino acid transport
gltA	b0720	gltA	ArcA(−), CRP(+), IHF(+)	+	GEA, IMP	[11]	tricarboxylic acid cycle, metabolic process, cellular carbohydrate metabolic process
grxB	b1064	grxB		+	GEA, IMP	[11]	cell redox homeostasis, oxidation-reduction process
hdeA	b3510	hdeAB- yhiD	FliZ(−), GadE(+), GadW(+/−), GadX(+/−), H-NS(−), Lrp(−), MarA(−), PhoP(+), RcsB(+), TorR(+)	+	MSI, GEA	[40]MSI, [13]GEA	cellular response to stress, cellular response to acidic pH
hdeB	b3509	hdeAB- yhiD	FliZ(−), GadE(+), GadW(+/−), GadX(+/−), H-NS(−), Lrp(−), MarA(−), PhoP(+), RcsB(+), TorR(+)	+	MSI	[10]	response to pH change, cellular response to stress
hdeD	b3511	hdeD	GadE(+), GadX(+), H-NS(−), PhoP(+),RcsB(+)	+	MSI	[10]	response to pH change
hdhA	b1619	hdhA		+	GEA, IMP	[11]	lipid metabolic process, metabolic process, steroid metabolic process, lipid catabolic process, bile acid, catabolic process, protein homotetramerization, oxidation-reduction process
iadA	b4328	yjiH G-iadA		+	MSI	[10]	proteolysis
luxS (ygaG)	b2687	luxS		+	MSI, GEA, IMP	[10]MSI, [11] GEA, IMP	cell-cell signaling involved in quorum sensing, L-methionine biosynthetic process from S-adenosylmethionine, quorum sensing
malE	b4034	malE FG	CRP(+), CreB(−), Fis(+), MalT(+)	+	GEA, IMP	[11]	cellular response to DNA damage stimulus, carbohydrate transport, maltose transport, detection of maltose stimulus, maltodextrin transport, cell chemotaxis
msrB	b1778	msrB		–	IMP	[13]	protein repair, response to oxidative stress
narU	b1469	narU		+	MSI	[10]	nitrate transport, nitrite transport, nitrate assimilation
ompF	b0929	ompF	CRP(+), CpxR(−), EnvY(+), Fur(+), IHF(+/−), OmpR(+/−), PhoB(+), RstA(−)	–	GEA, IMP	[41]	transport, ion transport, ion transport, drug transmembrane transport, bacteriocin transport
ompT	b0565	ompT envY -ompT	PhoP(+)	–	IMP	[13]	proteolysis
ompX	b1482	ompX	FNR(−)	+	GEA, IMP	[11]
osmC	b4376	osmC	H-NS(−), Lrp(+/−)	+	GEA, IMP	[11]	hyperosmotic response, response to oxidative stress, response to hydroperoxide, oxidation-reduction process
osmY	b1388	osmY	CRP(−), Fis(−), FliZ(−), IHF(−), Lrp(−)	+	GEA, IMP	[11]	response to osmotic stress
paaA	b1389	paaAB C D E F G H IJ K	CRP(+), IHF(+), PaaX(−)	+	MSI	[10]	phenylacetate catabolic process, oxidation-reduction process
paaB	b1391	paaAB C D E F G H IJ K	CRP (+), IHF (+), PaaX (−)	+	MSI	[10]	phenylacetate catabolic process
paaD	b1393	paaAB C D E F G H IJ K	CRP (+), IHF (+), PaaX (−)	+	MSI	[10]	phenylacetate catabolic process
paaF	b1395	paaAB C D E F G H IJ K	CRP (+), IHF (+), PaaX (−)	+	MSI	[10]	lipid metabolic process, fatty acid metabolic process, phenylacetate catabolic process
paaH	b1398	paaAB C D E F G H IJ K	CRP(+), IHF(+), PaaX(−)	+	MSI	[10]	fatty acid metabolic process, phenylacetate catabolic process, oxidation-reduction process
paaK	b3916	paaAB C D E F G H IJ K	CRP(+), IHF(+), PaaX(−)	+	MSI	[10]	metabolic process, phenylacetate catabolic process
pfkA	b0871	pfkA	Cra(−)	+	GEA, IMP	[11]	fructose 6-phosphate metabolic process, glycolytic process
poxB	b4226	poxB, poxB-l taE-ybjT	Cra(+), MarA(+), SoxS (+)	+	GEA, IMP	[11]	pyruvate metabolic process, oxidation-reduction process
ppa	b0384	ppa		+	GEA, IMP	[11]	phosphate-containing compound metabolic process
psiF	b1676	phoA -psiF	PhoB(+)	+	MSI	[10]
pykF	b1235	pykF	Cra(−)	+	GEA, IMP	[11]	glycolytic process, metabolic process, response to heat, phosphorylation
rssB	b0721	rssB		+	IMP	[10]	protein destabilization, positive regulation of proteolysis, regulation of nucleic acid-templated transcription (phosphorelay signal transduction system)
sdhC	b4719	sdhC DAB -sucA BC D	CRP(+), Fur(+), ArcA(+/−), Fnr(−)	–	IMP	[13]	aerobic respiration. Cytochrome complex assembly, tricarboxylic acid cycle, oxidation-reduction process
sdsN	b1646	sdsN		+	GEA	[42]	small RNA
sodC	b4059	sodC		+	GEA	[13]	superoxide metabolic process, removal of superoxide radicals, oxidation-reduction process
ssb	b0726	ssb	ArcA(−), LexA(−)	+	GEA, IMP	[11]	recombinational repair, DNA replication, cellular response to DNA damage stimulus, SOS response
sucA	b0729	sucA B sucA BC D	ArcA(+/−), FNR(−), IHF(−)	+	GEA, IMP	[11]	glycolytic process, tricarboxylic acid cycle, metabolic process, oxidation-reduction process
sucD	b2464	sucA B sucA BC D	ArcA(+/−),FNR(−),IHF(−)	+	GEA, IMP	[11]	tricarboxylic acid cycle, metabolic process, protein autophosphorylation
talA	b1886	talA- tktB	CreB(+)	+	GEA, IMP	[11]	carbohydrate metabolic process, pentose-phosphate shunt
tar	b1920	tar- tap-cheRBYZ	Fnr(+)	–	IMP	[13]	chemotaxis, signal transduction
tcyJ (fliY)	b3116	tcyJ fliA Z -tcyJ	H-NS(+), MatA(−), SutR(−), NsrR(−), CsgD(−), FlhDC(+)	–	IMP	[13]	L-cystine transport
tdcC	b3708	tdc AB C DEFG , tdc B C DEFG	CRP(+), FNR(+), IHF(+), TdcA(+), TdcR (+)	+	MSI	[10]	L-serine transport, threonine transport, proton transport, serine transport
tnaA	b3453	tna C A B	CRP(+), TorR (+)	+	GEA, IMP	[11]	cellular amino acid metabolic process, aromatic amino acid family metabolic process
ugpB	b3495	ugpB AECQ	CRP(+), PhoB(+/−)	+	GEA, IMP	[11]	glycerophosphodiester transport, transport, glycerol-3-phosphate transport
uspA	b0607	uspA	FadR(−), IHF(+)	+	GEA, IMP	[11]	response to stress
uspG (ybdQ)	b1004	uspG		+	GEA, IMP	[11]	response to stress, protein adenylylation, protein autophosphorylation, nucleotide phosphorylation, regulation of cell motility
wrbA	b0453	wrbA- yccJ	CsgD(+)	+	MSI, GEA, IMP	[13]MSI, [11] GEA, IMP	response to oxidative stress, negative regulation of transcription
ybaY	b0753	ybaY		+	MSI	[10]
ybgS	b0897	ybgS		+	MSI	[10]
ycaC	b1674	ycaC	BaeR(+), Fnr(−)	+	MSI, GEA, IMP	[10]MSI, [11] GEA, IMP	metabolic process
ydhY	b1784	ydhY VWXUT	FNR (+), NarL (−), NarP (−)	+	MSI	[10]	oxidation-reduction process
yeaH	b2013	yea G H	NtrC (+)	+	MSI	[10]
yeeE	b2665	yeeE D		+	MSI	[10]
ygaU	b3535	ygaU	CpxR (+)	+	GEA, IMP	[11]
yhjR	b3555	yhjR		+	MSI	[10]	bacterial cellulose biosynthetic process
yiaG	b4045	yiaG		+	MSI	[10]	regulation of transcription
yjbJ	b4329	yjbJ	FliZ (−)	+	MSI	[10]
yjiG	b1044	yjiH G-iadA		+	MSI	[10]
ymdA	b1138	ymdA		+	MSI	[10]
ymfE	b0885	ymfED		+	MSI	[10]

Genes regulated by Crl, Bnumbers, TUs to which they belong (in bold are possible candidates regulated by Crl, since they are controlled by Crl and σ^S, but they did not have a change of expression in the data we evaluated), TFs regulating the TU, the effect of Crl, evidences, references, and associated GO terms. Experimental evidence types supporting regulation by Crl: APPH Assay of protein purified to homogeneity, GEA Gene expression analysis, transcriptional fusions (lacZ), MSI Mapping of signal intensities, such as RNA-seq or microarray analysis; IMP Inferred from mutant phenotype (such as a mutation of a TF that has a visible cell phenotype and it is inferred that the regulator might be regulating the genes responsible for the phenotype). Growth conditions were 30 °C, as the stationary phase was induced for all experiments. All experiments were done with E. coli K-12 or derivative strains. This information, including regulatory interactions can be accessed at RegulonDB (http://regulondb.ccg.unam.mx/) by consulting the Crl regulon

Fig. 1.

Crl regulatory network in E. coli K-12. In green are those genes regulated positively and in red those regulated negatively. The regulatory effects of additional TFs are shown as green solid lines for activation and red solid lines for repression

Previously, genes under the control of Crl were classified in four main categories depending on their role(s) in the cell: DNA metabolism, central metabolism, response to environmental modifications, and miscellaneous [11]. Based on Gene Ontology (GO) annotations, multifunctional classification, and KEGG pathway maps to categorize functions, Crl-regulated genes appear to be involved in metabolic processes such as energy metabolism, amino acid, carbohydrate, and lipid metabolism, and biosynthetic processes such as glycan biosynthesis and biosynthesis of other secondary metabolites, among other metabolic processes. These functions correlate with results of the enrichment analysis using PANTHER, which showed that catabolic processes, metabolic processes, and cellular responses to xenobiotic stimuli were overrepresented among the functions associated with genes under the control of Crl (See Fig. 2).

Fig. 2.

Functions associated with genes under the control of Crl. GOs and Multifun-associated genes under Crl control and enrichment analysis with the PANTHER classification system and Multifun. Categories of KEGG used to classify GOs and Multifun terms are shown on the X-axis, and the number of GOs associated with each category are shown on the Y-axis

In general, genes under Crl control are involved in regulating many aspects of cellular metabolism through Crl’s interaction with a subset of genes of the σ^S regulon [8] in addition to quorum sensing playing a major role in cell-to-cell communication during stationary phase and in different processes such as biofilm formation or virulence, transporters [11] and genes involved in the uptake and utilization of β-glucosides [16].

Composition of the Crl regulon

In order to determine whether additional TFs also regulate the genes under the control of Crl, RegulonDB was used to evaluate how genes associated with Crl are also regulated by alternative TFs or sigma factors. A total of 24 genes were identified as exclusively controlled by Crl, whereas 62 are regulated by additional TFs (See Additional file 2). In this regard, 55 different TFs are involved in the regulation of genes associated with Crl, including Crp, IHF, H-NS, Fis, FNR, ArcA, GadX, GadW, GadE, and CsgD (Table 1), suggesting that all genes regulated by Crl are also involved in multiple functions beyond the stationary phase, or, alternatively, phase transition has to regulate genes involved in large number of different functions. It is interesting that six of seven global regulators identified in the regulatory network of E. coli are also associated with the set regulated by Crl. Another way to look at this small network is that 19 genes of the total of Crl-regulated genes are regulated by one TF, 11 by two TFs, and 14 by three different TFs. Therefore, Crl is regulating positively 73 (85%) genes, whereas 12 (15%) genes are regulated negatively (Table 1). The predominance of positive regulation suggests that genes associated with this regulon are in high demand according the demand theory suggested by Savageau [18], and the activities of their proteins are enhanced to contend with varied environmental stimuli. Thirty-four of the 86 genes have a σ^S-type promoter that was experimentally determined (RegulonDB). Finally, the promoters of 49 genes identified as members of Crl and of the σ^S sigmulon, based on transcriptional fusions and microarray analysis data, remain to be experimentally determined.

Phylogenetic analysis of Crl

In order to evaluate the phylogenetic history of Crl across the bacterial and archaeal cellular domains, its homologs were identified as described in the Methods section, and a phylogenetic tree with maximum likelihood was generated (Fig. 3 and Additional file 3). From this analysis, we found that Crl and its homologs are distributed almost exclusively among Gammaproteobacteria but do not share homology with proteins from other taxonomical divisions, as has been previously noted for E. coli, Vibrio spp., Citrobacter spp., Salmonella spp., and Enterobacter aerogenes [16]. Additional information suggests that Crl is less widespread and less conserved at the sequence level than σ^S [19]. In this regard, four conserved residues (Y22, F53, W56, and W82) are important for Crl activity and for Crl-σ^S interaction but not for Crl stability in S. Typhimurium [19]. On one hand it is probable that Crl homologs exist in some σ^S-containing bacteria; however, some species might use alternative strategies to favor σ^S interaction with the core of the RNAP [19]. Therefore, our phylogenetic analysis suggests that Crl is a protein conserved and constrained to Gammaproteobacteria, such as in Vibrio spp., Klebsiella spp., Enterobacter spp., and Escherichia coli. Contrary to Crl, several other of the TFs that co-regulate the Crl regulon, are present beyond the gamma-proteobacterial, probably pre-dating regulation of some of the target genes, which have been more recently subject to Crl regulation.

Fig. 3.

Phylogenetic tree of Crl. Phylogenetic tree based on Crl of E. coli and homologs in other organisms generated via maximum likelihood analysis, with 1000 replicates. Species with bootstrap values higher than 60% are displayed. The black triangles to the right of the branches indicate multiple species for those genera

In addition, homologous of Crl were found in low copy numbers, i.e., one Crl-like protein per genome. This information, together with the distribution of σ^S, suggests that the regulator was recruited as an element to regulate a subset of σ^S-regulated genes in Gammaproteobacteria. In this regard, it is interesting that genes under the control of Crl contain an UP element with A/T-rich DNA sequence upstream of a poorly conserved − 35 promoter which may serve for alpha subunit binding of RNAP; suggesting that Crl could play a fundamental role in the contacts between RNAP and its promoter [13]. In addition, Crl would increase transcription rate during the transition from growing to stationary phase at low temperatures [8], using indole as an effector molecule. In summary, this result opens the question explored in the follow section of whether genes regulated by Crl are also constrained to this taxonomical division.

Taxonomical distribution of Crl-regulated genes

Based on the identification of orthologs of 86 Crl-regulated genes, we evaluated their taxonomical distribution across archaea and bacteria sequence genomes, as described in Methods (See Fig. 4 and Additional file 4). Based on a taxonomical profile, we determined that the evolution of the Crl regulon seems to have involved diverse losses and gains of regulatory interactions. It is possible that large portions of the regulatory network associated with Crl evolved through extensive genetic changes during the evolution of the species studied. Indeed, we suggest three main events modeled the evolution of this regulon: (i) the regulation of a large number of genes widely distributed among Bacteria and Archaea, such as those genes involved in ancient metabolic processes such as glycolysis (fbaB, pykF, pfkA, and sucA) and those involved in the tricarboxylic acid cycle (gltA and sucD) [20]; (ii) the regulation of genes with a distribution pattern mainly constrained to Proteobacteria, with some secondary losses in other organisms, such as those genes involved in response to stress and starvation (cstA and hdcA) or cell adhesion (csgA and csgB), among others; and (iii) the recruitment of Crl as a consequence of its emergence in Enterobacteriales. It is interesting that Crl-regulated genes are also part of the σ^S sigmulon, where there are no essential genes [21–24]. All these elements suggest that the Crl regulon is highly flexible for phenotypic adaptation, probably as a consequence of the diverse growth environments associated with the organisms in which members of this regulatory network are present.

Fig. 4.

Taxonomic distribution of orthologs from the perspective of E. coli K-12. A single linkage-clustering algorithm with no leaf order optimization was applied with Pearson distance as the similarity measure. The display clustering results were obtained using the MeV program [39]. The conserved groups across the different taxonomic groups are indicated. Each column denotes Crl-regulated genes, whereas rows denote taxonomic groups. The bar at the top of the figure indicates the relative abundance of orthologs per group, represented as a percentage, where a value of 1 corresponds to 100% presence and 0% indicates a division without any ortholog of the Crl regulon in the taxonomic group

Conclusions

Crl stimulates -but can also repress- the association of σ^S with the RNAP core in E. coli K-12 through direct and specific interactions, increasing, or decreasing, the transcription rate of a subset of genes of the σ^S sigmulon. This TF has been described during the transition to stationary phase at low temperatures. In our work, based on an exhaustive literature search, we found 86 genes under the control of Crl in E. coli. We considered that the quality of the experiments could compensate the few number of papers where evidences associated to the rpoS-crl and its target genes; such as microarray analysis, mutations, assay with purified proteins, among others; however, a large number of records and sources were evaluated to consider the dataset as significant. Indeed, all interactions reported in this work meet the same criteria to be considered in RegulonDB. These protein-coding genes were retrieved mainly from microarray and mutation analyses, among other experimentally supported evidence. Gathering this regulon offers a wider physiological role than previously assumed for Crl. Certainly, these genes are associated with multiple functions, including xenobiotic processes, biofilm formation, metabolic, catabolic, and biosynthetic processes, responses to different stress conditions, and protein assembly, amino acid transport, and transcriptional processes, among others. The diverse functions regulated by Crl suggest that these genes play a fundamental role in multiple functions to respond to environmental changes, mainly those associated with stationary-phase growth at low temperatures. In addition, we conducted an exhaustive analysis concerning the conservation of the regulon Crl among the Bacteria and Archaea genomes, using as starting point, the knowledge gathered for E. coli K-12. From this analysis, Crl was identified in low copy numbers and constrained to the Enterobacteriales order, whereas the homologs of all regulated genes were found to be widely distributed beyond enterobacteria, suggesting that Crl was recruited in a secondary event to regulate a specific subset of genes for which the regulation (stimulation or repression) of Crl and σ^S helps bacteria in the phase transition.

Methods

Identification of Crl-regulated genes

We performed an exhaustive search of the literature related to Crl (GI: 114152792) in E. coli K-12 in PubMed [25] under the following search strategy: coli in the title (to exclude spurious articles) and crl, rpoS (ID: NP_417221) and regulation both in title and in all fields in their different combinations (coli[all field] crl[all field] rpos[all field] regulation[all field]; coli[ti] rpoS[ti] regulation[ti]; coli[ti] crl[all field] rpos[all field] regulation[all field]; coli[ti] crl[ti] regulation[all field]; coli[ti] rpoS[ti] crl[all field] regulation[all field]; coli[ti] rpoS[ti] crl[ti] regulation[all field]). Fifty nine unique articles with different search profiles were obtained and exhaustively revised, from which, 10 were selected since they contained information on regulatory interactions in E. coli on rpoS-crl and its target genes, such as microarray analysis, mutations, and assay with purified proteins, among others (Table 1). Finally, we searched for gene/operon notes in RegulonDB and EcoCyc [3, 26] for Crl interactions and σ^S promoters, for assembling the network of regulation of Crl.

The regulatory network generated was displayed using the Cytoscape program, version 3.3.0 [27], with information obtained in the identified papers as well as information contained in RegulonDB [3]. Genes under Crl control were classified based on Gene Ontology (GO) annotations (http://www.geneontology.org/) using the Gene Association Format (GAF 2.0) as well as the Multifun classification scheme [28]. An enrichment analysis was carried out to find overrepresented annotations, using the PANTHER Classification system program, version 12.0; selecting biological processes and E. coli as parameters [29, 30]. In addition, we used KEGG to categorize the functions of GOs (http://www.genome.jp/kegg-bin/show_organism?menu_type=pathway_maps&org=eco) [31].

Identification of Crl homologs

The Crl protein sequence of E. coli K-12 (ID: 114152792) was used as the seed to scan all the bacterial and archaeal genomes via a BLASTp search (BLAST version 2.2.30+) [32] (E-value ≤10^− 3 and coverage ≥60%). All proteins were compared and aligned using the Muscle algorithm [33] with default parameters, and results were manually edited with the program Jalview. Finally, a phylogeny was inferred by the maximum likelihood method with 1000 replicates by using the program MEGA [34] and the Tamura-Nei model.

Identification of orthologous genes

Orthologous genes have been classically defined as encoding proteins in different species that evolved from a common ancestor via speciation [35] and have retained the same function. In this work, orthologs were identified by searching for bidirectional best hits (BDBHs) in other organisms [36] considering the same conditions as [36] an E-value ≤ of 1e− 6; database size fixed (−z 5e+ 8), soft filtering of low information content (the −F ‘m S’ option), the Smith–Waterman alignment (−s T), and a coverage of at least 60%.

Taxonomical distribution of orthologous genes

In order to evaluate the taxonomical distribution of the genes belonging to the Crl regulon, 5321 complete genomes were downloaded from the NCBI’s Refseq genome database [37] and open reading frames (ORFs) that encode predicted proteins were considered. Redundancy was excluded using a web-based tool [38] considering a Genome Similarity Score GCCa≥0.95 [38]. In this representative genome dataset, orthologs were traced along 18 archaeal and 69 bacterial cellular divisions. To this end, the relative abundance of the orthologs was calculated as the fraction of genomes in the group that had one ortholog, divided by the total number of genomes per phylum, i.e., the ratio (total number of orthologs in a phylum) / (total number of organisms in phylum). The corresponding matrix was analyzed with a hierarchical complete linkage-clustering algorithm with correlation uncentered as the similarity measure. We used the program MeV to perform the analyses (http://mev.tm4.org/) [39].

Abbreviations

APPH

Assay of protein purified to homogeneity

BDBHs

Bidirectional best hits

BLASTp

Basic Local Alignment Search Tool (proteins)

Crl

Curling genes regulatory protein

Ecocyc

E. coli metabolic database

GAF

cGMP-specific phosphodiesterase, adenylyl cyclase and FhlA domain

GCCa

Genome Similarity Score

GEA

Gene expression analysis, transcriptional fusions (lacZ)

GO

Gene onthology

IMP

Inferred from mutant phenotype (such as a mutation of a TF that has a visible cell phenotype and it is inferred that the regulator might be regulating the genes responsible for the phenotype)

Jalview

Java alignment editor

KEGG

Kyoto encyclopedia of genes and genomes database

MEGA

Molecular Evolutionary Genetics Analysis

MSI

Mapping of signal intensities, such as RNA-seq or microarray analysis

Muscle

Multiple sequence comparison by Log-expectation

ORFs

Open reading frames

PANTHER

Protein analysis through evolutionary relationships classification system

PhoPQ

Two-component system of virulence and adaptation

RegulonDB

Regulon database

RNAP

RNA polymerase

RNA-seq

RNA sequencing

RpoD

RNA polymerase sigma factor D

RpoS

RNA polymerase sigma factor S

TFs

Transcription factors

TUs

Transcription units

Authors’ contributions

ASZ, JCV and EPR conceived and designed the research. EPR and ASZ designed and drafted the manuscript, contributed equally in the development of this work and they are the major contributors in writing the manuscript. MSP and EPR performed the bioinformatics analysis. MSP and DAVR gathered the regulation of the network. ASZ and DAVR contributed to the data extraction, and curation of the processed data of the articles. EPR performed the phylogenetic analyzes. All authors read and approved the final manuscript.

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

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