Bacterial Small Rnas in the Genus Herbaspirillum Spp

International Journal of Molecular Sciences

Article Bacterial Small RNAs in the Genus Herbaspirillum spp.

Amanda Carvalho Garcia 1,* , Vera Lúcia Pereira dos Santos 1, Teresa Cristina Santos Cavalcanti 2, Luiz Martins Collaço 2 and Hans Graf 1

1 Department of Internal Medicine, Federal University of Paraná, Curitiba 80.060-240, Brazil; [email protected] (V.L.P.d.S.); [email protected] (H.G.) 2 Department of Pathology, Federal University of Paraná, PR, Curitiba 80.060-240, Brazil; [email protected] (T.C.S.C.); [email protected] (L.M.C.) * Correspondence: [email protected]; Tel.: +55-41-99833-0124

Received: 5 November 2018; Accepted: 12 December 2018; Published: 22 December 2018

Abstract: The genus Herbaspirillum includes several strains isolated from different grasses. The identification of non-coding RNAs (ncRNAs) in the genus Herbaspirillum is an important stage studying the interaction of these molecules and the way they modulate physiological responses of different mechanisms, through RNA–RNA interaction or RNA–protein interaction. This interaction with their target occurs through the perfect pairing of short sequences (cis-encoded ncRNAs) or by the partial pairing of short sequences (trans-encoded ncRNAs). However, the companion Hfq can stabilize interactions in the trans-acting class. In addition, there are Riboswitches, located at the 50 end of mRNA and less often at the 30 end, which respond to environmental signals, high temperatures, or small binder molecules. Recently, CRISPR (clustered regularly interspaced palindromic repeats), in prokaryotes, have been described that consist of serial repeats of base sequences (spacer DNA) resulting from a previous exposure to exogenous plasmids or bacteriophages. We identified 285 ncRNAs in Herbaspirillum seropedicae (H. seropedicae) SmR1, expressed in different experimental conditions of RNA-seq material, classified as cis-encoded ncRNAs or trans-encoded ncRNAs and detected RNA riboswitch domains and CRISPR sequences. The results provide a better understanding of the participation of this type of RNA in the regulation of the metabolism of bacteria of the genus Herbaspirillum spp.

Keywords: RNA non-coding (ncRNA); ncRNA cis-encoded; trans-encoded; riboswitches; CRISPR; mRNA

1. Introduction The genus Herbaspirillum was described by Baldani et al. [1] and includes the Herbaspirillum seropedicae (H. seropedicae) species with several strains isolated from different grasses [1]. Herbaspirillum seropedicae is characterized by fixing nitrogen (diazotrophic) and colonizing plants. A second diazotrophic species, Pseudomonas rubrisubalbicans, was reclassified under the name of Herbaspirillum rubrisubalbicans and also has the ability to colonize plants [2]. From the 1990s, new species were included in the genus Herbaspirillum: H. autotrophicum (syn. Aquaspirillum autotrophicum) and H. huttiense (syn. Pseudomonas huttiense)[3] H. frisingense [4], H. lusitanum [5], H. cholorophenolicum [6], H. hiltneri [7], H. rhizosphaerae [8], H. aquaticum [9], H. massiliense [10], H. canariense, H. aurantiacum, and H. soli [11], and H. seropedicae AU14040 [12]. The H. putei species was reclassified as H. huttiense subspecies putei [9]. H. seropedicae belongs to the beta class of Proteobacteria, is gram-negative, and displays a characteristic growth pattern in semi-solid culture medium and without nitrogen [1,13]. It is a

Int. J. Mol. Sci. 2019, 20, 46; doi:10.3390/ijms20010046 www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2019, 20, 46 2 of 20 bacterium that colonizes intracellular spaces and the xylem of grass roots, such as sugarcane and sorghum [1,14]; and intracellular spaces in seeds and rice roots [14,15]. It can also be isolated from the stem leaves of various grasses [15–17]. The genome of the H. seropedicae strain (SmR1) has been completely sequenced and annotated by the GENOPAR (Paraná Genome) consortium. “Wordiness 4,735” Open Reading frames (ORFs) occupy about 88.3% of the genome, on a single circular chromosome. In this annotation, the genes of non-coding RNAs (apart from rRNAs and tRNAs), are not included. Gene encoding regulatory RNAs, known as small RNAs (sRNAs) or non-coding sRNAs (ncRNAs), are transcribed in trans and in cis relative to target RNA [18,19]. The work of the researchers Anderson et al. were the first to isolate and characterize the RNA encoded by the micF gene in Escherichia coli [20]. The genes are located between the coding regions for the proteins, that is, in the intergenic regions of the genome and display signals from the promoters and terminator sequences that are usually Rho-independent [21–23]. The size of the ncRNAs genes ranges from ~50 to ~500 nucleotides and several transcripts were processed by RNase into smaller products [24–27]. They modulate physiological responses through different mechanisms, by RNA-RNA interaction or RNA-protein interactions, and some interactions can be regulated by the Hfq chaperone [28–30]. The most studied ncRNAs are the cis-encoded RNAs and the trans-encoded RNAs. The cis-encoded RNAs are transcribed in cis and encoded at the same locus as their target mRNA in the antisense sense duplex structure, in addition to having a post-transcriptional gene regulation of responses and a high degree of sequence complementarity, which was considered to be an indication that interaction of the Hfq protein [31,32] would not be required. However, some investigators reported interference of Hfq in the ncRNA, mRNA targeting, and cis-encoded target [33–35]. Overall, these ncRNAs act by complementing the mRNA ribosome-binding site, as well as inhibiting the conversion [33,36]. In contrast, trans-encoded ncRNAs that are encoded in trans, have target mRNAs at different sites in the genome where the formation of the ncRNA occurs: the Hfq—mRNA complex may act positively or negatively on post-transcriptional regulation [37]. This is due to the imperfect base pairing which prevents their eventual degradation by RNase [38,39]. The regulation of gene expression of trans-encoded ncRNAs was first discovered by Dalihas, who described the micF and dicF found in Escherichia coli, and the lin-4 found in the nematode Caenorhabditis elegans, which are encoded by genes located at different sites of their target genes and only display a partial complementarity with their target RNAs [40]. The Riboswitches, which are located in the 50 UTR region of an mRNA, constitute another class of ncRNAs and lead to transcriptional regulation through their interaction with a linker molecule [41–43]. However, the presence of small, non-coding RNAs in different Herbaspirillum species remains undetermined. A total number of 285 ncRANs are needed to predict the novel candidate sRNAs within the intergenic regions (Herbaspirillum seropedicae SmR1). We further analyzed the predicted ncRNAs in great detail in Herbaspirillum spp. using Infernal 1.1.1 bioinformatic tools. Moreover, these analyses constitute the very first documented evidence of the presence of sRNAs in Herbaspirillum genomes.

2. Results

2.1. Prediction of New ncRNAs in the Genus Herbaspirillum spp. The computational tool Infernal 1.1.1 was employed for this study (see Section 4.3) and the multiple covariance detection and sequence analysis had as its input the RNA family database (Rfam, available link http://rfam.xfam.org/; accessed on 18 February 2016), while the prediction of the 16 species of ncRNAs made up the genus Herbaspirillum spp., as shown in Supplementary Material S1. The complete list of genomes is as follows: H. seropedicae Z67, H. lusitanum P6-12, H. hiltneri N3, H. frisingense GSF30, Herbaspirillum spp. strain B65, H. rubrisubalbicans M1, H. autotrophicum Int. J. Mol. Sci. 2019, 20, 46 3 of 20

IAM 14942, H. huttiense subsp. putei, Herbaspirillum spp. strain B501, Herbaspirillum spp. strain GW103, Herbaspirillum spp. strain RV423, H. rhizosphaerae UMS-37, Herbaspirillum spp. strain Os34, Herbaspirillum spp. strain Os45, and H. seropedicae AU14040. The results are listed in Table1. By adopting this approach, no ncRNAs were detected in H. lusitanum P6-12 and Herbaspirillum spp. strain B65, due to the signiﬁcant number of scaffolds and contigs that are identiﬁed in incomplete genomes. The organisms with the highest number of common ncRNAs were H. seropedicae SmR1, H. seropedicae Z67, H. hiltneri N3, H. frisingense, H. rubrisubalbicans M1, and H. seropedicae AU14040, shown in Supplementary Material S4.

Table 1. ncRNAs predicted for bacteria of the genus Herbaspirillum and different from those already known in H. seropedicae SmR1. The Infernal (version 1.1.1) computational tool was used.

Genus Herbaspirillum ncRNAs Herbaspirillum seropedicae Z67 ohsC_RNA, Rhizobiales-2 and UPD-PKc Herbaspirillum lusitanum P6-12 Absence Herbaspirillum hiltneri N3 ar14, Acido-1, Alpha_RBS, ar14, asX2, asX3, Bp2_287, csRNA, isrN, istR, Ms_AS-4, Ms_IGR-8, ncr1175, NsiR1, P26, P5, PrrB_RsmZ, RsmY, RydC, sau-5971, SpF41_sRNA, Spot_42, Xoo5, sraA, sX9, TB10Cs4H2, TB11Cs5H2 and TB9Cs1H1 Herbaspirillum frisingense GSF30 Entero_5_CRE, P10 and STnc430 Herbaspirillum spp. estirpe B65 snoZ248 Herbaspirillum rubrisubalbicans M1 ALIL, Alpha_RBS, Archaea_SRP, asX2, b55, BjrC1505, C4, ceN84, istR, K_chan_RES, MAT2A_B, MEG3_2, NRF2_IRES, OrzO-P, P36, pRNA, PtaRNA1, rli60, rox2, rseX, sau-6072, sau-63, SpF25_sRNA, SpR10_sRNA, sroB, sX15, TB9Cs1H1 and TB9Cs1H2. Herbaspirillum huttiense subsp. Putei estirpe IAM asX2, Cardiovirus_CRE, Chloroﬂexi-1, MEG3_2, 15032 SBWMV2_UPD-PKk, sroB, veev_FSE, asX2, Cardiovirus_CRE, Chloroﬂexi-1, MEG3_2, SBWMV2_UPD-PKk, sroB and veev_FSE. Herbaspirillum autotrophicum IAM 14942 MIR1444 and SpF10_sRNA. Herbaspirillum spp. estirpe B501 Absence Herbaspirillum spp. estirpe GW103 sX2, Flavi_CRE, RyeB, SAM-IV, sau-6072 e sroB. Herbaspirillum spp. estirpe RV1423 psRNA2, sbcD and STnc40. Herbaspirillum rhizosphaerae UMS-37 NRF2_IRES, NsiR1, Pseudomon-groES and sX9. Herbaspirillum rubrisubalbicans spp. estirpe Os34 ryfA, SpR20_sRNA and asX2. Herbaspirillum rubrisubalbicans spp. estirpe Os45 asX2, ROSE_2 and ryfA. Genus Herbaspirillum = 15 species; ncRNAs = non-coding RNAs.

The ctRNA_p42d, Betaproteobacteria_toxic_sRNA, and P10 ncRNAs are examples of ncRNAs found in Herbaspirillum spp. RV423, H. rhizosphaerae UMS-37, and Herbaspirillum spp. OS34 and O45 and common to H. seropedicae SmR1. The ncRNAs Betaproteobacteria_toxic_sRNA, pﬂ, SX4, and alpha_tmRNA, AR35, tmRNA, among others, are repeated in all the species, except for Herbaspirillum spp. B65, Herbaspirillum spp. B501, and Herbaspirillum lusitanum P6-12. The characterization of the ncRNAs predicted in the genus Herbaspirillum spp. suggests that despite the differences within these species, there is sequence preservation among the common ncRNAs. In Herbaspirillum seropedicae SmR1, the prediction of ncRNAs resulted in 108 ncRNAs, as shown in Figure1. The sequence of ncRNAs predicted in H. seropedicae SmR1 by Infernal 1.1.1 is shown in Supplementary Material S2. All ncRNAs predicted by the Infernal 1.1.1 tool in the genus Herbaspirillum spp. are shown in Supplementary Material S4. Int. J. Mol. Sci. 2018, 19, x FOR PEER REVIEW 3 of 19

Z67, H. hiltneri N3, H. frisingense, H. rubrisubalbicans M1, and H. seropedicae AU14040, shown in Supplementary Material S4. The ctRNA_p42d, Betaproteobacteria_toxic_sRNA, and P10 ncRNAs are examples of ncRNAs found in Herbaspirillum spp. RV423, H. rhizosphaerae UMS-37, and Herbaspirillum spp. OS34 and O45 and common to H. seropedicae SmR1. The ncRNAs Betaproteobacteria_toxic_sRNA, pfl, SX4, and alpha_tmRNA, AR35, tmRNA, among others, are repeated in all the species, except for Herbaspirillum spp. B65, Herbaspirillum spp. B501, and Herbaspirillum lusitanum P6-12. The characterization of the ncRNAs predicted in the genus Herbaspirillum spp. suggests that despite the differences within these species, there is sequence preservation among the common ncRNAs. In Herbaspirillum seropedicae SmR1, the prediction of ncRNAs resulted in 108 ncRNAs, as shown in Int.Figure J. Mol. 1. Sci. The2019 sequence, 20, 46 of ncRNAs predicted in H. seropedicae SmR1 by Infernal 1.1.1 is shown4 of in20 Supplementary Material S2.

Int. J. Mol. Sci. 2018, 19, x FOR PEER REVIEW 4 of 19

All ncRNAs predicted by the Infernal 1.1.1 tool in the genus Herbaspirillum spp. are shown in Supplementary Material S4.

2.2. ncRNAs Differential Expression in RNA-Seq Data from RNA-seq data were used in 11 culture conditions that were tested in Herbaspirillum seropedicae SmR1, as described in Supplementary Material S3. Less than five covering values were includedFigure 1. and Examples 1. these were ofExamples the regarded cis-encoded of as 5possible′ the_ureB_sRNA,cis predicted-encoded rimP, ncRNAs. PyrR, 50_ureB_sRNA, CsrC, It was Betaproteobacteria_toxic_sRNA, observed rimP, that PyrR, there were CsrC, ncRNAs expressedBetaproteobacteria_toxic_sRNA,riboswitch, in FMN,all 11 cobalamin,conditions, ykkC-yxkD, but others riboswitch, sucA; were andonly FMN, the expressed trans cobalamin,-encoded in some sX4, ykkC-yxkD, of sX11, the sX6,conditions. sucA;MicC, IsrK, and IsrD, the transIsrG.The -encodedThe 5′_ureB_sRNA CRISPR sX4, RNA sX11, region in sX6, H. predicted MicC,seropedicae IsrK,the H. SmR1 IsrD, seropedicae IsrG. has in 286 The the nucleotides CRISPRSmR1 genome. RNA and region is classified predicted as the cisH.-encoded antisenseseropedicae ncRNAin the SmR1depending genome. on its function and localization of the genome, is incorporated with the Table 1. ncRNAs predicted for bacteria of the genus Herbaspirillum and different from those already 2.2.ureA, ncRNAs ureB, Differential ureC, ureD, Expression ureE, andin ureF RNA-Seq (sense) genes, is expression the NFbHPN-Malate-Naringenin and NFbHP-Malate-2known in H. seropedicae conditions, SmR1. asThe shown Infernal in (version Figure 1.1.1)2. H. computationalseropedicae SmR1 tool wasuses used. naringenin as a carbon sourceDataGenus and from Herbaspirillum is found RNA-seq in the data plant, were where used the in biosynthesis 11 culture ofncRNAs flavonoids conditions occurs; that were its compounds tested in are HerbaspirilluminvolvedHerbaspirillum in seropedicaethe seropedicae plant defense Z67SmR1, mechanism. as described in Supplementary ohsC_RNA, Rhizobiales-2 Material and S3. UPD-PKc Less than five covering valuesHerbaspirillum wereThe ncRNA included lusitanum 5′_ureB_sRNA and P6-12 these were is repeated regarded in some as possible genera of predicted AbsenceHerbaspirillum ncRNAs. by having It was 100% observed sequence thatidentity thereHerbaspirillum were such ncRNAsas, hiltneri for example, N3 expressed in ar14,H. in seropedicae allAcido-1, 11 conditions, Alpha_RBS, Z67, H. ar14,lusitanum but asX2, others asX3,, H. were hiltneriBp2_287, only N3, csRNA, expressed H. rubrisubalbicansisrN, istR, in someMs_AS- of M1, 4, Ms_IGR-8, ncr1175, NsiR1, P26, P5, PrrB_RsmZ, RsmY, RydC, sau-5971, the conditions. and H. seropedicae AU14040. TheSpF41_sRNA, location of Spot_42, the ureABC Xoo5, sraA, operon sX9, TB10Cs4H2,locus in the TB11Cs5H2 genome and of TB9Cs1H1these species 0 wasHerbaspirillumThe identified. 5 _ureB_sRNA frisingense The presence GSF30 in H. seropedicae of ureaseCSmR1 (alpha), has ureaseA 286 Entero_5_CRE, nucleotides (gamma), P10 and and and STnc430 is ureaseB classified (beta) as cis subunits-encoded was antisensedetectedHerbaspirillum ncRNA and it spp. was depending estirpe found B65 that on itsthe function 5′-ureaB andsRNA localization ncRNAs were of snoZ248the found genome, in these is incorporated species of Herbaspirillum with the ureA,interactsHerbaspirillum ureB, ureC,in cisrubrisubalbicans ureD,of the ureE,ureaseC M1and subunit.ureF (sense) ALIL, The Alpha_RBS, ncRNA genes, mRNA is Archaea_SRP, expression 5′ UTR asX2, the cspA NFbHPN-Malate-Naringeninb55, is BjrC1505, a thermoregulator C4, ceN84, istR, which has and NFbHP-Malate-2 conditions,K_chan_RES, as shown inMAT2A_B, Figure2 MEG3_2,. H. seropedicae NRF2_IRES,SmR1 OrzO-P, uses P36, naringeninpRNA, PtaRNA1, as a 373 nucleotides in H. seropedicae SmR1.rli60, rox2, It is rseX, classified sau-6072, as sau-63, a cis SpF25_sRNA,-encoded ncRNA, SpR10_sRNA, which sroB, appears sX15, as a carbonmRNA source 5′ mRNA and is sequence found in theof the plant, gene where annotated the biosynthesis as cspDTB9Cs1H1 (Hsero_1397 of and flavonoids TB9Cs1H2.). In addition, occurs; its there compounds is a second areHerbaspirillumcspD involved gene in( huttienseHsero_3028 the plant subsp.) defense that Putei does mechanism. notasX2, contain Cardiovirus_CRE, the mRNA Chloroflexi sequence-1, MEG3_2,5′ UTR. SBWMV2_UPD-PKk, sroB, estirpe IAM 15032 veev_FSE, asX2, Cardiovirus_CRE, Chloroflexi-1, MEG3_2, SBWMV2_UPD- PKk, sroB and veev_FSE. Herbaspirillum autotrophicum IAM MIR1444 and SpF10_sRNA. 14942 Herbaspirillum spp. estirpe B501 Absence Herbaspirillum spp. estirpe GW103 sX2, Flavi_CRE, RyeB, SAM-IV, sau-6072 e sroB. Herbaspirillum spp. estirpe RV1423 psRNA2, sbcD and STnc40. Herbaspirillum rhizosphaerae UMS-37 NRF2_IRES, NsiR1, Pseudomon-groES and sX9. Herbaspirillum rubrisubalbicans spp. ryfA, SpR20_sRNA and asX2. estirpe Os34 Herbaspirillum rubrisubalbicans spp. asX2, ROSE_2 and ryfA. estirpe Os45 Genus Herbaspirillum = 15 species; ncRNAs = non-coding RNAs.

(a)

Figure 2. Cont.

Int. J. Mol. Sci. 2019, 20, 46 5 of 20 Int. J. Mol. Sci. 2018, 19, x FOR PEER REVIEW 5 of 19

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Figure 2. Visualization of position and expression profile of 5_ureB_sRNA ncRNA in the genome of Figure 2. Visualization of position and expression profile of 5_ureB_sRNA ncRNA in the genome of H. H. seropedicae SmR1. The sequence was located using the Artemis program. The readings delineated seropedicae SmR1. The sequence was located using the Artemis program. The readings delineated within the within the pink rectangle refer to: (a) Levels of expression of 5_ureB_sRNA ncRNA in RNA-seq assays pink rectangle refer to: (a) Levels of expression of 5_ureB_sRNA ncRNA in RNA-seq assays in H. seropedicae in H. seropedicae SmR1. In color, the analyzed experimental conditions represented are shown, where 1 SmR1. In color, the analyzed experimental conditions represented are shown, where 1 and 2 represent d.a.i and 2 represent d.a.i (days after inoculation); (b) the numbers represent the value of the gene expression (days after inoculation); (b) the numbers represent the value of the gene expression in reads per kilobase of in reads per kilobase of transcript per million mapped reads (RPKM). transcript per million mapped reads (RPKM). The ncRNA 50_ureB_sRNA is repeated in some genera of Herbaspirillum by having 100% sequence identity suchYamanaka as, for example, and Inouye in H. seropedicae [44] used Z67, the H. cspD-lacZ lusitanum fusion, H. hiltneri and foundN3, H. rubrisubalbicansthat the cspD expressionM1, and H.induced seropedicae byAU14040. stationary The phase location growth of the does ureABC not depend operon locus on sigma in the factor genome 6S. of Thus, these the species cspD gene was identified.expression The is presenceinversely of dependent ureaseC (alpha), on the ureaseA growth, (gamma), rates induced and ureaseB by glucose (beta) starvation. subunits wasThey also detectedobserved and it was that found the 5′ that UTR the cspA 50-ureaB mRNA sRNA is expressed ncRNAs were in the found all assay in these conditions species of andHerbaspirillum showed a higher interactsexpression in cis of the in ureaseC seq RNA subunit. assays The at ncRNA NFbHPN-Malate-Nitrate mRNA 50 UTR cspA and is a NFbHP-Malate-Nitrate-2thermoregulator which and has 373NFbHPN-Malate-High-Oxygen-1, nucleotides in H. seropedicae SmR1. as It is shown classified in Figure as a cis -encoded 3. NFbHPN-Malate-Nitrate-1 ncRNA, which appears is as where a H. mRNAseropedicae 50 mRNA SmR1 sequence rises of to the 30 gene°C in annotatedNFbHP medium, as cspD using(Hsero_1397 malate). as In a addition,carbon source there and is a 10 second mM nitrate cspD geneas a (nitrogenHsero_3028 source) that [45]. does not contain the mRNA sequence 50 UTR. Yamanaka and Inouye [44] used the cspD-lacZ fusion and found that the cspD expression induced by stationary phase growth does not depend on sigma factor 6S. Thus, the cspD gene expression is inversely dependent on the growth, rates induced by glucose starvation. They also observed that the 50 UTR cspA mRNA is expressed in the all assay conditions and showed a higher expression in seq RNA assays at NFbHPN-Malate-Nitrate and NFbHP-Malate-Nitrate-2 and NFbHPN-Malate-High-Oxygen-1, as shown in Figure3. NFbHPN-Malate-Nitrate-1 is where H. seropedicae SmR1 rises to 30 ◦C in NFbHP medium, using malate as a carbon source and 10 mM nitrate as a nitrogen source [45].

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Int. J. Mol. Sci. 2018, 19, x FOR PEER REVIEW 5 of 19

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Figure 2. Visualization of position and expression profile of 5_ureB_sRNA ncRNA in the genome of H. seropedicae SmR1. The sequence was located using the Artemis program. The readings delineated within the pink rectangle refer to: (a) Levels of expression of 5_ureB_sRNA ncRNA in RNA-seq assays in H. seropedicae SmR1. In color, the analyzed experimental conditions represented are shown, where 1 and 2 represent d.a.i (days after inoculation); (b) the numbers represent the value of the gene expression in reads per kilobase of transcript per million mapped reads (RPKM).

Yamanaka and Inouye [44] used the cspD-lacZ fusion and found that the cspD expression induced by stationary phase growth does not depend on sigma factor 6S. Thus, the cspD gene expression is inversely dependent on the growth, rates induced by glucose starvation. They also observed that the 5′ UTR cspA mRNA is expressed in the all assay conditions and showed a higher expression in seq RNA assays at NFbHPN-Malate-Nitrate and NFbHP-Malate-Nitrate-2 and NFbHPN-Malate-High-Oxygen-1, as shown in Figure 3. NFbHPN-Malate-Nitrate-1 is where H.

Int.seropedicae J. Mol. Sci. 2019 SmR1, 20, 46rises to 30 °C in NFbHP medium, using malate as a carbon source and 10 mM6 nitrate of 20 as a nitrogen source [45].

Int. J. Mol. Sci. 2018, 19, x FOR PEER REVIEW 6 of 19 (a)

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0 FigureFigure 3. 3Visualization. Visualization of of the the position position and and expression expression proﬁle profile of of mRNA mRNA 5 5′UTR UTRcspA cspAin in the the genome genome ofof H.H. seropedicaeseropedicaeSmR1. SmR1. TheThe sequencesequence waswas locatedlocated withwith thethe aidaidthe theArtemis Artemisprogram. program. TheThe readingsreadings 0 delineateddelineated within the pink pink rectangle rectangle refer refer to: to: (a) ( aLevels) Levels of expression of expression of mRNA of mRNA 5′ UTR 5 UTRcspA incspA RNA-in RNA-seqseq assays assays in H. inseropedicaeH. seropedicae SmR1.SmR1. In the In colored the colored part, part, the the11 analyzed 11 analyzed experimental experimental conditions conditions are arerepresented, represented, where where 1 and 1 and 2 2represent represent d.a.i d.a.i (days (days after after inoculation); inoculation); ( (bb)) the numbersnumbers represent represent thethe valuevalue ofof thethe genegene expressionexpression in in RPKM. RPKM.

TheThe ncRNAncRNA sX4 has 113 113 nucleotides nucleotides and and is is classified classiﬁed as as transtrans-encoded-encoded antisense antisense ncRNA. ncRNA. It is It co- is co-locatedlocated with with the the ampGampG, tufB, tufB, and, and trnGtrnG genes.genes. The The designation designation sX4 sX4 refers refers to the ncRNAs describeddescribed inin XanthomonasXanthomonas campestris campestris pv. pv. vesicatoria vesicatoria[46 [46].]. In InH. H. seropedicae seropedicaeSmR1, SmR1, the the sX4 sX4 ncRNA ncRNA was was expressed expressed in all in theall the tested tested conditions, conditions, but but the highestthe highest expression expression in Adhered-corn-3-days in Adhered-corn-3-days conditions, conditions, followed followed by the by Plankton-corn-3-days-2.the Plankton-corn-3-days-2. It was Itfound was that found the greatest that the expression greatest occurred expression during occurred the colonization during the of maize,colonization as shown of maize, in Figure as4 shown. Thus, itin shouldFigure be4. notedThus, thatit should the greatest be noted expression that the occurred greatest duringexpression the colonizationoccurred during of corn. the colonization of corn.

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Figure 3. Visualization of the position and expression profile of mRNA 5′ UTR cspA in the genome of H. seropedicae SmR1. The sequence was located with the aid the Artemis program. The readings delineated within the pink rectangle refer to: (a) Levels of expression of mRNA 5′ UTR cspA in RNA- seq assays in H. seropedicae SmR1. In the colored part, the 11 analyzed experimental conditions are represented, where 1 and 2 represent d.a.i (days after inoculation); (b) the numbers represent the value of the gene expression in RPKM.

The ncRNA sX4 has 113 nucleotides and is classified as trans-encoded antisense ncRNA. It is co- located with the ampG, tufB, and trnG genes. The designation sX4 refers to the ncRNAs described in Xanthomonas campestris pv. vesicatoria [46]. In H. seropedicae SmR1, the sX4 ncRNA was expressed in all the tested conditions, but the highest expression in Adhered-corn-3-days conditions, followed by the Plankton-corn-3-days-2. It was found that the greatest expression occurred during the Int.colonization J. Mol. Sci. 2019 of, 20maize,, 46 as shown in Figure 4. Thus, it should be noted that the greatest expression7 of 20 occurred during the colonization of corn.

Int. J. Mol. Sci. 2018, 19, x FOR PEER REVIEW 7 of 19 (a)

(b) Figure 4. Visualization of the position and expression proﬁle of the sX4 ncRNA in the genome Figure 4. Visualization of the position and expression profile of the sX4 ncRNA in the genome of H. of H. seropedicae SmR1. The sequence was located by means the Artemis program. The readings seropedicae SmR1. The sequence was located by means the Artemis program. The readings delineated delineated by the pink rectangle are as follows: (a) Levels of sX4 ncRNA expression in RNA-seq assays by the pink rectangle are as follows: (a) Levels of sX4 ncRNA expression in RNA-seq assays in H. in H. seropedicae SmR1. In the colored section, the 11 analyzed experimental conditions are represented, seropedicae SmR1. In the colored section, the 11 analyzed experimental conditions are represented, where 1 and 2 represent d.a.i (days after inoculation); (b) the numbers represent the value of the gene where 1 and 2 represent d.a.i (days after inoculation); (b) the numbers represent the value of the gene expression in RPKM. expression in RPKM. In H. seropedicae the cobalamin riboswitch has 113 nucleotides and is classiﬁed as trans-encoded antisenseInncRNA H. seropedicae or cobalamin the cobalamin element. riboswitch It is co-located has 113 with nucleotides the Hsero_2659 and geneis classified (cobalt transporter)as trans-encoded as a sequenceantisense 5 0ncRNAUTR, and or cobalamin with the genes element.Hsero_2660 It is co-locatedand Hsero_2661 with the(antisense Hsero_2659 sense) gene genes. (cobalt Coenzyme transporter) B12as or a cobalamin sequence riboswitches 5′ UTR, and are with control the elements genes Hsero_2660 that are widespread and Hsero_2661 in prokaryotes. (antisense For sense) example, genes. theCoenzymemetE gene B12 contains or cobalamin a cobalamin riboswitchesriboswitch areand control binding elements of coenzyme that are widespread B12 to this elementin prokaryotes. that leadsFor to example, the formation the metE of agene transcriptional contains a terminatorcobalamin andriboswitch the repression and binding of metE of coenzymeexpression. B12 In to the this absenceelement of the that coenzyme, leads to the metE formationmRNA of is a synthesized transcriptional [47]. NFbHPN-Malate-Low-Oxygen-1terminator and the repression of and metE NFbHPN-Malate-Naringenin-1expression. In the absence of arethe shown coenzyme, in Figure the metE5. These mRNA results is synthesized indicate that [47]. there NFbHPN-Malate- is likely to be noLow-Oxygen-1 binding of the CbtBand NFbHPN-Malate-Naringenin-1 (cobalt carrier subunit) linker to are the shownhsero_2659 in FiguremRNA 5. These in the results aptamer indicate region, that wherethere an is alternate likely to clampbe no binding loader formed. of the CbtB (cobalt carrier subunit) linker to the hsero_2659 mRNA in the aptamer region, where an alternate clamp loader formed.

(a)

Int. J. Mol. Sci. 2018, 19, x FOR PEER REVIEW 7 of 19

(b)

Figure 4. Visualization of the position and expression profile of the sX4 ncRNA in the genome of H. seropedicae SmR1. The sequence was located by means the Artemis program. The readings delineated by the pink rectangle are as follows: (a) Levels of sX4 ncRNA expression in RNA-seq assays in H. seropedicae SmR1. In the colored section, the 11 analyzed experimental conditions are represented, where 1 and 2 represent d.a.i (days after inoculation); (b) the numbers represent the value of the gene expression in RPKM.

In H. seropedicae the cobalamin riboswitch has 113 nucleotides and is classified as trans-encoded antisense ncRNA or cobalamin element. It is co-located with the Hsero_2659 gene (cobalt transporter) as a sequence 5′ UTR, and with the genes Hsero_2660 and Hsero_2661 (antisense sense) genes. Coenzyme B12 or cobalamin riboswitches are control elements that are widespread in prokaryotes. For example, the metE gene contains a cobalamin riboswitch and binding of coenzyme B12 to this element that leads to the formation of a transcriptional terminator and the repression of metE expression. In the absence of the coenzyme, the metE mRNA is synthesized [47]. NFbHPN-Malate- Low-Oxygen-1 and NFbHPN-Malate-Naringenin-1 are shown in Figure 5. These results indicate that Int.there J. Mol. is likely Sci. 2019 to, 20 be, 46 no binding of the CbtB (cobalt carrier subunit) linker to the hsero_2659 mRNA8 of 20in the aptamer region, where an alternate clamp loader formed.

Int. J. Mol. Sci. 2018, 19, x FOR PEER REVIEW 8 of 19 (a)

(b) Figure 5. Visualization of the position and expression profile of the cobalamin ncRNA in the Figure 5. Visualization of the position and expression profile of the cobalamin ncRNA in the H. H. seropedicae genome SmR1. The sequence was located by means of the Artemis program. The readings seropedicae genome SmR1. The sequence was located by means of the Artemis program. The readings are delineated by the pink rectangle (a). Levels of ncRNA cobalamin expression in RNA-seq assays are delineated by the pink rectangle (a). Levels of ncRNA cobalamin expression in RNA-seq assays in in H. seropedicae SmR1. In the colored section, the 11 analyzed experimental conditions represented, H. seropedicae SmR1. In the colored section, the 11 analyzed experimental conditions represented, where 1 and 2 represent d.a.i (days after inoculation). (b) The numbers represent the value of the gene where 1 and 2 represent d.a.i (days after inoculation). (b) The numbers represent the value of the gene expression in RPKM. expression in RPKM. Non-coding RNAs were regarded as essential (housekeeping ncRNAs), such as 6S ncRNA, and were alsoNon-coding identified RNAs by the were Infernal regarded 1.1.1 as tool. essential In Herbaspirillum (housekeeping seropedicae ncRNAs), SmR1,such as mcRNA6S ncRNA, 6S and has were a sequencealso identified of 177 nucleotides by the Infernal and expression 1.1.1 tool. inIn RNA-seqHerbaspirillum material. seropedicae The ncRNA SmR1, 6S mcRNA in E. coli 6Sis has responsible a sequence forof regulating 177 nucleotides the activity and of expression the RNA inpolymerase RNA-seq (RNAP) material. that The is ncRNA dependent 6S in on E. the coli sigma is responsible factor 70, for andregulating necessary the for activity the recognition of the RNA of the polymerase promoter (RNAP) and initiation that is dependent of the transcription. on the sigma In addition, factor 70, it and globallynecessary regulates for the the recognition gene expression of the responsepromoter to and growth initiation from of the the exponential transcription. phase In addition, to the stationary it globally phaseregulates [48]. It the is significant gene expression that the response level of to expression growth from was the found exponential to be high phase relative to the to itsstationary neighbors phase (Hsero_1224[48]. It isand significantHsero_1225 that) under the level the conditions of expression tested, was and found this confirms to be highthat this relative ncRNA to is its essential. neighbors In addition,(Hsero_1224 the and 6S Hsero_1225 ncRNA was) under expressed the conditions in all the tested, conditions and this tested, confirms with that an this emphasis ncRNA is on essential. the Adhered-wheat-1In addition, the condition,6S ncRNA aswas shown expressed in Figure in all6 .the conditions tested, with an emphasis on the Adhered- wheat-1 condition, as shown in Figure 6.

(a)

Int. J. Mol. Sci. 2018, 19, x FOR PEER REVIEW 8 of 19

(b)

Figure 5. Visualization of the position and expression profile of the cobalamin ncRNA in the H. seropedicae genome SmR1. The sequence was located by means of the Artemis program. The readings are delineated by the pink rectangle (a). Levels of ncRNA cobalamin expression in RNA-seq assays in H. seropedicae SmR1. In the colored section, the 11 analyzed experimental conditions represented, where 1 and 2 represent d.a.i (days after inoculation). (b) The numbers represent the value of the gene expression in RPKM.

Non-coding RNAs were regarded as essential (housekeeping ncRNAs), such as 6S ncRNA, and were also identified by the Infernal 1.1.1 tool. In Herbaspirillum seropedicae SmR1, mcRNA 6S has a sequence of 177 nucleotides and expression in RNA-seq material. The ncRNA 6S in E. coli is responsible for regulating the activity of the RNA polymerase (RNAP) that is dependent on the sigma factor 70, and necessary for the recognition of the promoter and initiation of the transcription. In addition, it globally regulates the gene expression response to growth from the exponential phase to the stationary phase [48]. It is significant that the level of expression was found to be high relative to its neighbors (Hsero_1224 and Hsero_1225) under the conditions tested, and this confirms that this ncRNA is essential. Int.In J.addition, Mol. Sci. 2019 the, 6S20, ncRNA 46 was expressed in all the conditions tested, with an emphasis on the Adhered-9 of 20 wheat-1 condition, as shown in Figure 6.

Int. J. Mol. Sci. 2018, 19, x FOR PEER REVIEW 9 of 19 (a)

(b)

FigureFigure 6. The 6. The sequence sequence was was located located by meansby means ofthe of the Artemis Artemis program. program. The The readings readings delineated delineated by theby the pinkpink rectangle rectangle were were as follows: as follows: (a) Visualization (a) Visualization of expression of expression levels levels of ncRNA of ncRNA 6S in 6S RNA-seq in RNA-seq assays assays in H. seropedicaein H. seropedicaeSmR1. SmR1. In the coloredIn the colored section, section, the 11 analyzed the 11 analyzed experimental experimental conditions conditions represented, represented, where 1 andwhere 2 show 1 and d.a.i 2 (daysshow afterd.a.i inoculation);(days after inoculation); (b) the numbers (b) the represent numbers the represent value of the the gene value expression of the gene in RPKM.expression in RPKM.

2.3.2.3. Comparative Comparative Analysis Analysis of theof the Secondary Secondary Structure Structure of 6S of of6S H. of seropedicaeH. seropedicae SmR1 SmR1 GivenGiven the theimportance importance of this of ncRNA, this ncRNA, there was there a comparative was a comparative analysis of analysis the secondary of the structure secondary of thestructure 6S of H. of seropedicae the 6S of SmR1,H. seropedicae together SmR1, with the together 6S of withJanthinobacterium the 6S of Janthinobacteriumspp. and Burkholderia spp. and CCGE1003.Burkholderia The CCGE1003. analysis was The conducted analysis with was the conducted RNAFold with [49 ] the tool, RNAFold and based [49] on tool, thermodynamic and based on RNA-RNAthermodynamic interactions, RNA-RNA where the interactions, secondary where structures the of secondary nCRNA 6S structures were generated. of nCRNA The 6S three were structuresgenerated. shown The three in Figure structures7 are basicallyshown in similar Figure (a7 are long basically rod shown similar in (a Figure long 7rodB for shown the 6Sin ofFigure Janthinobacterium7B for the 6S ofspp.). Janthinobacterium However, there spp. are). However, variations inthere the presenceare variations and number in the presence of loop out and structures number of thatloop may out or structures may not have that amay functional or may meaning.not have a A functional multiple sequence meaning. alignment A multiple was sequence carried alignment out by thewas CLUSTALW carried out tool by [50 the], as CLUSTALW shown in Figure tool8 .[50], It is observedas shown that in Figure there is 8. a differenceIt is observed in the that secondary there is a structuredifference of ncRNA in the secondary 6S between structure these species, of ncRNA even if6S they between present these a high species, degree even of conservationif they present in a the high aligneddegree nucleotide of conservation sequence. in the aligned nucleotide sequence.

Figure 7. Visualization of the secondary structure of ncRNA 6S. (a) H. seropedicae SmR1; (b) Janthinobacterium sp.; (c) Burkholderia CCGE1003. A predictive analysis of the secondary structures of identified ncRNAs was conducted by means of RNAFold.

Int. J. Mol. Sci. 2018, 19, x FOR PEER REVIEW 9 of 19

(b)

Figure 6. The sequence was located by means of the Artemis program. The readings delineated by the pink rectangle were as follows: (a) Visualization of expression levels of ncRNA 6S in RNA-seq assays in H. seropedicae SmR1. In the colored section, the 11 analyzed experimental conditions represented, where 1 and 2 show d.a.i (days after inoculation); (b) the numbers represent the value of the gene expression in RPKM.

2.3. Comparative Analysis of the Secondary Structure of 6S of H. seropedicae SmR1 Given the importance of this ncRNA, there was a comparative analysis of the secondary structure of the 6S of H. seropedicae SmR1, together with the 6S of Janthinobacterium spp. and Burkholderia CCGE1003. The analysis was conducted with the RNAFold [49] tool, and based on thermodynamic RNA-RNA interactions, where the secondary structures of nCRNA 6S were generated. The three structures shown in Figure 7 are basically similar (a long rod shown in Figure 7B for the 6S of Janthinobacterium spp.). However, there are variations in the presence and number of loop out structures that may or may not have a functional meaning. A multiple sequence alignment was carried out by the CLUSTALW tool [50], as shown in Figure 8. It is observed that there is a Int.difference J. Mol. Sci. in2019 the, 20 secondary, 46 structure of ncRNA 6S between these species, even if they present10 a ofhigh 20 degree of conservation in the aligned nucleotide sequence.

Figure 7. Visualization of the secondary structure of ncRNA 6S. (a) H. seropedicae SmR1; (bFigure) Janthinobacterium 7. Visualization sp.; ( ofc) Burkholderia the secondary CCGE1003. structure A of predictive ncRNA analysis 6S. (a) ofH. the seropedicae secondary structures SmR1; (b) Int. J. Mol. Sci. 2018, 19, x FOR PEER REVIEW 10 of 19 ofJanthinobacterium identiﬁed ncRNAs sp.; was (c) conducted Burkholderia by meansCCGE1003. of RNAFold. A predictive analysis of the secondary structures Int. J. Mol. Sci. 2018, 19, x FOR PEER REVIEW 10 of 19 of identified ncRNAs was conducted by means of RNAFold.

FigureFigure 8. 8.MultipleMultiple alignment alignment of 6S ncRNA of 6S ncRNA gene sequences gene sequences by the CLUSTALW by the CLUSTALW tool. Hsero = tool. Herbaspirillum Hsero = seropedicaeHerbaspirillumFigure 8. MultipleSmR1; seropedicae Janthinobacteri alignmentSmR1; of 6S = Janthinobacteri JanthinobacteriumncRNA gene sequences = Janthinobacterium sp.; and by Burkholderia the CLUSTALWsp.; and = Burkholderia Burkholderia tool. Hsero CCGE1003. = =HerbaspirillumBurkholderia CCGE1003.seropedicae SmR1; Janthinobacteri = Janthinobacterium sp.; and Burkholderia = Burkholderia CCGE1003. 2.4. Candidate sRNA Target Identification 2.4. Candidate sRNA Target Identiﬁcation 2.4. InCandidate H. seropedicae sRNA TargetSmR1, Identification the consensus ribosomal binding site (RBS)sequence and its variations In H. seropedicae SmR1, the consensus ribosomal binding site (RBS)sequence and its variations were Inexamined, H. seropedicae since SmR1,this is thethe consensusRBS AGGA ribosomal nucleus andbinding the siteCAAGGACA (RBS)sequence sequence. and its Take variations as an were examined, since this is the RBS AGGA nucleus and the CAAGGACA sequence. Take as an examplewere examined, the nucleotide since thissequence is the ofRBS ncHSmR1_04, AGGA nucleus which and has the 40 CAAGGACA possible targets. sequence. They include Take as the an example the nucleotide sequence of ncHSmR1_04, which has 40 possible targets. They include the serS serSexample gene thatthe nucleotideencodes a seryl-tRNA sequence of synthetase. ncHSmR1_04, which has 40 possible targets. They include the gene that encodes a seryl-tRNA synthetase. serSIt gene is worth that encodesnoting that a seryl-tRNA the base pairing synthetase. between the ncHSmR1_04 and the mRNA serS, occurs in It is worth noting that the base pairing between the ncHSmR1_04 and the mRNA serS, occurs in the RBSIt is region worth of noting the latter, that the as shownbase pairing in Figure between 9. This the is ncHSmR1_04 a strong indication and the that mRNA the ncHSmR1_04serS, occurs in the RBS region of the latter, as shown in Figure9. This is a strong indication that the ncHSmR1_04 negativelythe RBS region regulate of thethe latter,conversion as shown of seryl-tRNA in Figure 9.synthetase. This is a strongThe 4.5S indication RNA is athat common the ncHSmR1_04 ncRNA in negatively regulate the conversion of seryl-tRNA synthetase. The 4.5S RNA is a common ncRNA bacterianegatively and regulate its purpose the conversion is to the function of seryl-tRNA of directing synthetase. proteins The containing4.5S RNA is signal a common peptide ncRNA to the in in bacteria and its purpose is to the function of directing proteins containing signal peptide to the secretorybacteria andapparatus, its purpose and thus is to form the afunction signal-recognizing of directing particle proteins with containing the Fhh signalprotein. peptide In addition, to the secretory apparatus, and thus form a signal-recognizing particle with the Fhh protein. In addition, the thesecretory 4.5S RNA apparatus, takes part and in thusthe conversation form a signal-recognizing when interacting particle with thewith G the stretch Fhh factorprotein. of GIn [51].addition, 4.5S RNA takes part in the conversation when interacting with the G stretch factor of G [51]. the 4.5S RNA takes part in the conversation when interacting with the G stretch factor of G [51].

Figure 9. It is worth noting that the base pairing between the ncHSmR1_04 and the mRNA serS occurs inFigureFigure the RBS 9.9. It region is worth of the noting latter. that the base pairing between the ncHSmR1_04 andand thethe mRNAmRNA serSserS occursoccurs inin thethe RBSRBS regionregion ofof thethe latter.latter. In E. coli, the 4.5S RNA is encoded by the gene (ffs) and contains 114 nucleotides. It is the bifunctionalIn E. coli molecule, the 4.5S that RNA is involvedis encoded in by the the conversion gene (ffs ) and and secretion contains of 114 protein nucleotides. by binding It is the to elongationbifunctional factor molecule (EFG) and that Ffh is involvedthe protein in respectively the conversion [52]. Apparently, and secretion the of 4.5S protein RNA byfacilitates binding the to releaseelongation of the factor EF-G-GTP (EFG) andfrom Ffh the the ribosome protein respectivelyby competing [52]. with Apparently, the 23S rRNA the 4.5S for RNA binding facilitates with the the EF-Grelease [51,53]. of the EF-G-GTP from the ribosome by competing with the 23S rRNA for binding with the EF-GBuskiewicz [51,53]. et al. [54] analyzed the functions of the SRP (signal-recognizing particle) in E. coli that wereBuskiewicz based on et theal. [54]binding analyzed of the the Fth functions protein to of the the 4.5 SRP S RNA(signal-recognizing and the Fth-4.5 particle) S complex. in E. The coli researchersthat were based suggested on the that binding the free of Fth, the theFth 4.5Sprotein RNA, to andthe 4.5FtsY S bindingRNA and sites the are Fth-4.5 occluded S complex. by strong The domain–domainresearchers suggested interactions that the that free must Fth, be the disrupted 4.5S RNA, by and the FtsYSRP orbinding Fth-FtsY sites complex are occluded formation. by strong domain–domainThe 4.5S RNA interactions of H. seropedicae that must SmR1 be disruptedcontains 146 by thenucleotides. SRP or Fth-FtsY Among complex the targets formation. of 4.5S, as predictedThe by4.5S Target RNA RNA2, of H. seropedicaeis the mutL SmR1 mRNA contains that encodes 146 nucleotides. a protein involved Among in the DNA targets repair. of Again,4.5S, as thepredicted matching by ofTarget the ncRNA RNA2, occursis the mutL close mRNA to that thatof the encodes RBS of athe protein target involved RNA and in in DNA this repair.specific Again, case, startsthe matching at the third of the base ncRNA upstream occurs of theclose adenosine to that of of the the RBS AUG of thecodon, target as RNAshown and in inFigure this specific10. case, starts at the third base upstream of the adenosine of the AUG codon, as shown in Figure 10.

Figure 10. It is worth noting that the base pairing between the 4.5S and the mRNA mutL start at the thirdFigure base 10. upstream It is worth of notingthe adenosine that the of base the pairingAUG codon. between the 4.5S and the mRNA mutL start at the third base upstream of the adenosine of the AUG codon.

Int. J. Mol. Sci. 2018, 19, x FOR PEER REVIEW 10 of 19

Figure 8. Multiple alignment of 6S ncRNA gene sequences by the CLUSTALW tool. Hsero = Herbaspirillum seropedicae SmR1; Janthinobacteri = Janthinobacterium sp.; and Burkholderia = Burkholderia CCGE1003.

2.4. Candidate sRNA Target Identification In H. seropedicae SmR1, the consensus ribosomal binding site (RBS)sequence and its variations were examined, since this is the RBS AGGA nucleus and the CAAGGACA sequence. Take as an example the nucleotide sequence of ncHSmR1_04, which has 40 possible targets. They include the serS gene that encodes a seryl-tRNA synthetase. It is worth noting that the base pairing between the ncHSmR1_04 and the mRNA serS, occurs in the RBS region of the latter, as shown in Figure 9. This is a strong indication that the ncHSmR1_04 negatively regulate the conversion of seryl-tRNA synthetase. The 4.5S RNA is a common ncRNA in bacteria and its purpose is to the function of directing proteins containing signal peptide to the secretory apparatus, and thus form a signal-recognizing particle with the Fhh protein. In addition, the 4.5S RNA takes part in the conversation when interacting with the G stretch factor of G [51].

Int. J. Mol.Figure Sci. 20199. It ,is20 worth, 46 noting that the base pairing between the ncHSmR1_04 and the mRNA serS occurs11 of 20 in the RBS region of the latter.

InIn E.E. coli coli,, thethe 4.5S4.5S RNARNA isis encodedencoded byby thethe gene gene ( ffs(ffs)) and and contains contains 114 114 nucleotides. nucleotides. It It is is the the bifunctionalbifunctional molecule molecule that that is is involved involved in in the the conversion conversion and and secretion secretion of of protein protein by by binding binding to to elongationelongation factor factor (EFG)(EFG) andand FfhFfh thethe proteinprotein respectivelyrespectively [[52].52]. Apparently,Apparently, the the 4.5S 4.5S RNARNA facilitatesfacilitates thethe releaserelease ofof thethe EF-G-GTPEF-G-GTP fromfrom thethe ribosomeribosome byby competingcompeting withwith thethe 23S23S rRNArRNA forfor bindingbinding withwith thethe EF-GEF-G [[51,53].51,53]. BuskiewiczBuskiewicz etet al.al. [[54]54] analyzedanalyzed thethe functionsfunctions ofof thethe SRPSRP (signal-recognizing(signal-recognizing particle)particle) inin E.E. colicoli thatthat werewere basedbased onon thethe bindingbinding ofof thethe FthFth proteinprotein toto the the 4.5 4.5 S S RNA RNA and and the the Fth-4.5 Fth-4.5 S S complex. complex. TheThe researchersresearchers suggestedsuggested thatthat thethe freefree Fth,Fth, thethe 4.5S4.5S RNA,RNA, andand FtsYFtsY bindingbinding sitessites areare occludedoccluded byby strongstrong domain–domaindomain–domainInt. J. Mol. Sci. 2018 interactionsinteractions, 19, x FOR PEER thatthat REVIEW mustmust be be disrupted disrupted by by the the SRP SRP or or Fth-FtsY Fth-FtsY complex complex formation. formation. 11 of 19 TheThe 4.5S4.5S RNARNA ofofH. H. seropedicaeseropedicaeSmR1 SmR1contains contains146 146nucleotides. nucleotides. AmongAmong thethe targetstargets ofof 4.5S,4.5S, asas The analysis of the ncRNA 4.5S conducted by the TargetRNA2 tool resulted in 77 target mRNAs predictedpredicted by by TargetTarget RNA2, RNA2, is is the themutL mutLmRNA mRNA thatthat encodesencodes aa proteinprotein involvedinvolved inin DNADNA repair.repair. Again,Again, in this set and includes the nifQ gene whose product is involved in nitrogen metabolism, as shown thethe matchingmatching ofof thethe ncRNAncRNA occursoccurs closeclose toto thatthat ofof thethe RBSRBS ofof thethe targettarget RNARNA andand inin thisthis speciﬁcspecific case,case, in Figure 11. startsstarts atat thethe thirdthird base base upstream upstream of of the the adenosine adenosine of of the the AUG AUG codon, codon, as as shown shown in in Figure Figure 10 10..

FigureFigureFigure 10.10. 11.It It is isIt worth worthis worth noting noting noting that that that the the basethe base base pairing pairing pairing between between betweenthe the 4.5S the4.5S 4.5S and and theand the mRNA mRNAthe mRNAmutL mutL nifQ startstart predicted atat the the pairing starts 21 bases upstream of the AUG codon, and hence is outside of the RBS region. Int. J. Mol.thirdthird Sci. base base 2018 upstream upstream, 19, x FOR of ofPEER the the adenosine REVIEWadenosine of of the the AUG AUG codon. codon. 11 of 19

It can be seen that the predicted pairing starts 21 bases upstream of the AUG codon, and hence The analysis of of the the ncRNA ncRNA 4.5S 4.5S conducted conducted by by the the TargetRNA2 TargetRNA2 tool resulted in 77 target target mRNAs mRNAs is outside of the RBS region, as shown in Figure 12. This means that according to TargetRNA2, when inin this set andand includesincludes thethe nifQnifQgene gene whose whose product product is is involved involved in in nitrogen nitrogen metabolism, metabolism, as as shown shown in compared with the previous example, an interaction energy value is obtained for the pairing: −16.21 inFigure Figure 11 .11. for mutL and −8.31 for nifQ. These two factors (the pairing position and interaction energy value) make nifQ an unlikely target for 4.5S.

2.5. Prediction of CRISPR

A new category of non-coding RNA, CRISPR RNA, has been described for both the Bacteria Figure 11. ItIt is is worth worth noting noting that that the the base base pairing between the the 4.5S 4.5S and the mRNA nifQ predicted domain and the Archaea domain [55,56]. The CRISPR transcription factor results in small fragments pairing starts 21 bases upstream of the AUG codon, and hence is outside of the RBS region. of RNA that recognize a specific exogenous DNA and that can guide the Cas nuclease, that is responsibleItIt can be seen for the that cleavage thethe predictedpredicted of this pairing pairingDNA if starts startsit re-contacts 21 21 bases bases upstreamthe upstream microorganism. of of the the AUG AUG Thus, codon, codon, prokaryotes and and hence hence have is isoutside outsidea defense of of the mechanismthe RBS RBS region, region, against as as shown shown invasive in in Figure FigureDNAs 12 12.[57].. ThisThis In meansthismeans study, thatthat the accordingaccording CRISPRFinder to TargetRNA2, and CRISPRmap when comparedtools were with used the previous to predict example, CRISPR an in interaction the genome energyenergy of H. valuevalue seropedicae isis obtainedobtained SmR1. forfor A thethe CRISPR pairing:pairing: locus −−16.2116.21 was forforidentified mutL and with−8.31−8.31 164for for nifQnifQ nucleotides. .These These two two divided factors factors into (the (the DRpairing pairing (54 position positionnucleotides) and and interaction interaction and SPA (57energy energy nucleotides) value) value) makepalindromic nifQ an unlikely repetitions, target as for shown 4.5S. in Figure 12.

2.5. Prediction of CRISPR A new category of non-coding RNA, CRISPR RNA, has been described for both the Bacteria domain and the Archaea domain [55,56]. The CRISPR transcription factor results in small fragments of RNAFigureFigure that 12. 12. recognizeVisualization Visualization a specificof theof the CRISPR CRISPR exogenous locus locus in DNA thein the genome genome and that of H.of canH.seropedicae seropedicae guideSmR1. the SmR1. Cas Sense Sense nuclease, sense sense in thein that the is responsiblegenome,genome, for with thewith 164 cleavage 164 nucleotides nucleotides of this divided dividedDNA into if intoit DR re-contacts DR (54 (54 nucleotides) nucleotides) the microorganism. palindromic palindromic repetitions Thus,repetitions prokaryotes and and spacing spacing have a defenseregionregion mechanism (57 (57 nucleotides) nucleotides) against underlined. underlined. invasive DNAs [57]. In this study, the CRISPRFinder and CRISPRmap tools were used to predict CRISPR in the genome of H. seropedicae SmR1. A CRISPR locus was 2.5. Prediction of CRISPR identifiedThe withCRISPR 164 locus nucleotides is co-located divided with the into rhtB DR gene (54 (sense-sense) nucleotides) and and with SPA the psiF (57 and nucleotides) Hsero_1878 palindromicgenesA new (in the categoryrepetitions, antisense-sense), of as non-coding shown as inshown RNA,Figure in CRISPR 12.Figure 13. RNA, With has regard been to described this, CRISPR for is both expressed the Bacteria in some domainconditions, and the with Archaea the highest domain level [55 being,56]. The expression CRISPR in transcription the condition factor NFbHPN-Malato-Alto-Oxygen-1 results in small fragments of a RNAcondition that recognize where H. a speciﬁcseropedicae exogenous SmR1 is DNA using and malate that canas a guide carbon the source Cas nuclease, up to OD that of is 0.4. responsible This result forsuggests the cleavage that the of expression this DNA ifof it the re-contacts CRISPR locus the microorganism.in the NFbHPN-Malate-High-Oxygen-1 Thus, prokaryotes have condition a defense acts mechanismas a signal against for the invasivenitrogen DNAssource [57that]. Inthe this bacterium study, the is using, CRISPRFinder by assisting and in CRISPRmap controlling the tools pH were of the usedmedium, to predict since CRISPR the psiF in gene the is genome expressed of H. during seropedicae the phosphateSmR1. A deprivation. CRISPR locus was identiﬁed with Figure 12. Visualization of the CRISPR locus in the genome of H. seropedicae SmR1. Sense sense in the genome, with 164 nucleotides divided into DR (54 nucleotides) palindromic repetitions and spacing region (57 nucleotides) underlined.

The CRISPR locus is co-located with the rhtB gene (sense-sense) and with the psiF and Hsero_1878 genes (in the antisense-sense), as shown in Figure 13. With regard to this, CRISPR is expressed in some conditions, with the highest level being expression in the condition NFbHPN-Malato-Alto-Oxygen-1 a condition where H. seropedicae SmR1 is using malate as a carbon source up to OD of 0.4. This result suggests that the expression of the CRISPR locus in the NFbHPN-Malate-High-Oxygen-1 condition acts as a(a) signal for the nitrogen source that the bacterium is using, by assisting in controlling the pH of the medium, since the psiF gene is expressed during the phosphate deprivation.

(a)

Int. J. Mol. Sci. 2018, 19, x FOR PEER REVIEW 11 of 19

The analysis of the ncRNA 4.5S conducted by the TargetRNA2 tool resulted in 77 target mRNAs in this set and includes the nifQ gene whose product is involved in nitrogen metabolism, as shown in Figure 11.

Figure 11. It is worth noting that the base pairing between the 4.5S and the mRNA nifQ predicted pairing starts 21 bases upstream of the AUG codon, and hence is outside of the RBS region.

It can be seen that the predicted pairing starts 21 bases upstream of the AUG codon, and hence is outside of the RBS region, as shown in Figure 12. This means that according to TargetRNA2, when compared with the previous example, an interaction energy value is obtained for the pairing: −16.21 for mutL and −8.31 for nifQ. These two factors (the pairing position and interaction energy value) make nifQ an unlikely target for 4.5S.

2.5. Prediction of CRISPR A new category of non-coding RNA, CRISPR RNA, has been described for both the Bacteria domain and the Archaea domain [55,56]. The CRISPR transcription factor results in small fragments of RNA that recognize a specific exogenous DNA and that can guide the Cas nuclease, that is responsible for the cleavage of this DNA if it re-contacts the microorganism. Thus, prokaryotes have a defense mechanism against invasive DNAs [57]. In this study, the CRISPRFinder and CRISPRmap tools were used to predict CRISPR in the genome of H. seropedicae SmR1. A CRISPR locus was identified with 164 nucleotides divided into DR (54 nucleotides) and SPA (57 nucleotides) palindromic repetitions, as shown in Figure 12.

Int. J. Mol. Sci. 2019, 20, 46 12 of 20 Figure 12. Visualization of the CRISPR locus in the genome of H. seropedicae SmR1. Sense sense in the genome, with 164 nucleotides divided into DR (54 nucleotides) palindromic repetitions and spacing 164 nucleotidesregion (57 divided nucleotides) into DR underlined. (54 nucleotides) and SPA (57 nucleotides) palindromic repetitions, as shown in Figure 12. The CRISPRThe CRISPR locus locus is co-located is co-located with with the rhtB the rhtBgene gene (sense-sense) (sense-sense) and withand with the psiF the psiFand Hsero_1878and Hsero_1878 genesgenes (in the (in antisense-sense), the antisense-sense), as shown as shown in Figure in Figure 13. With 13. With regard regard to this, to this, CRISPR CRISPR is expressed is expressed in some in some conditions,conditions, with with the highestthe highest level level being being expression expression in the in conditionthe condition NFbHPN-Malato-Alto-Oxygen-1 NFbHPN-Malato-Alto-Oxygen-1 a a conditioncondition where whereH. seropedicae H. seropedicaeSmR1 SmR1 is using is using malate malate as a as carbon a carbon source source upto up OD to ofOD 0.4. of This0.4. This result result suggestssuggests that that the expressionthe expression of theof the CRISPR CRISPR locus locus in in the the NFbHPN-Malate-High-Oxygen-1 NFbHPN-Malate-High-Oxygen-1 conditioncondition acts acts as a signal for thethe nitrogen sourcesource thatthat thethe bacteriumbacterium isis using,using, byby assistingassisting inin controllingcontrolling thethe pHpH ofof the the medium,medium, sincesince thethe psiFpsiF genegene is expressed during the phosphate phosphate deprivation. deprivation.

Int. J. Mol. Sci. 2018, 19, x FOR PEER REVIEW 12 of 19 (a)

(b)

FigureFigure 13. 13. Visualization of the position of CRISPR in the the genome genome of of H.H. seropedicae seropedicae SmR1 SmR1 ( (a).a). VisualizationVisualization of of expression expression levels levels of of CRISPR CRISPR in in RNA-seq RNA-seq assays assays in inH. H. seropedicae seropedicaeSmR1. SmR1. In In the the colored colored section,section, the the 11 11 analyzed analyzed experimental experimental conditions conditions areare represented, represented, where where 1 1 and and 2 2 represent represent d.a.i d.a.i (days (days afterafter inoculation); inoculation); ( b(b)) the the numbers numbers represent represent the the value value of of the the gene gene expression expression in in RPKM. RPKM.

InIn addition, addition, the the CRISPRFinder CRISPRFinder tool tool identified identified the the CRISPR-associated CRISPR-associated protein, protein, while while the the Cmr4 Cmr4 FamilyFamily RAMP in in H.H. seropedicae seropedicae SmR1SmR1 interacted interacted with with casRAMP_Cmr4, casRAMP_Cmr4, which which participates participates in the process in the processof developing of developing immunological immunological e memory. e memory.The Cas RAMP The Cas complex RAMP modulates complex modulates between the between cleavage the of cleavagethe RNAs of [58–61]. the RNAs The [58 results–61]. Thesuggest results that suggest the CRISPR-Cas that the CRISPR-Cas system is a systemprokaryotic is a prokaryotic defense mechanism defense mechanismagainst foreign against genetic foreign elements. genetic The elements. main features The main of this features defense of thissystem defense are Cas system proteins are Cas and proteins CRISPR andRNA. CRISPR However, RNA. since However, the different since thenitrogen different sources nitrogen have sourcesvariations have in variations the amount in of the nitrogen amount and of nitrogenCaCo3 equivalents, and CaCo3 acidification equivalents, and acidification the reduction and theof soil reduction pH can ofbecome soil pH significant. can become significant.

Int. J. Mol. Sci. 2019, 20, 46 13 of 20

3. Discussion In the RNA-seq assays, the highest expression of 5_ureB_sRNA ncRNA in the NFbHPN-Malate-Naringenin condition was observed, which may suggest that this 5_ureB_sRNA ncRNA is involved in the regulation of flavonoid biosynthesis. These act as a protection against oxidizing agents in the plant and involve the use of externally generated and internally generated urea as a source of carbon. Urea is metabolized by urease, a nickel-dependent metalloenzyme, which catalyzes the hydrolysis of urea to form ammonia and carbon dioxide, and can be found in bacteria, fungi, and plants. [62]. In Klebsiella aerogenes, the ureABC operon encodes the three subunits of the pimple enzyme [62]. In Helicobacter pylori, the urease cluster consists of two operons (ureAB and ureIEFGH). This bacterium is the only one described to date that contains the 5_ureB_sRNA, an antisense cis-encoded ncRNA with 290 nucleotides, which negatively regulates ureAB expression when paired at the 50-region of the ureB mRNA [63]. H. seropedicae SmR1 has a probable ureABC operon and a 50_ureB ncRNA, with 286 nucleotides. According to the prediction of Infernal 1.1.1, this ncRNA is a cis-encoded antisense that is positioned close to the ureC gene. This difference can be explained by the fact that (a) H. pylori does not have the ureC gene, (b) the H. seropedicae SmR1 gene sequences are larger than those of H. pylori, and (c) the metabolism of urea involves different proteins. According to Yamanaka and Inouye [44], in E. coli there are new homologues of cspA, denominated cspA to cspI, and the cspD gene does not have the 50 UTR sequence. This information suggests that in H. seropedicae SmR1, the genes cspD (Hsero_1397) is actually the cspA gene. This gene is co-located with the genes rsuA, icd, clpS, clpA (sense sense). Jiang et al. [43] analyzed the function of the unconverted 50 UTR sequence CspA mRNA from the major cold shock adaptation protein to CspA in E. coli. The researchers observed that adaptation to cold shock is blocked when the 50 UTR CspA mRNA region is overexpressed, through the synthesis not only of the CspA protein, but also of proteins from the CspA family such as CspB, CspC, CspD, and CspE. The cspD gene in E. coli codes for a protein of homologous sequence with the cold shock protein to the CspA, but the expression of cspD is not induced by the cold shock [44]. The sX4 ncRNA whose expression is dependent on the HrpG and HrpX proteins, suggests that the target (s) of this ncRNA can be found in the interaction of the bacterium with the plant [46,64]. Studies with HrpG and HrpX mutants in X. campestris pv. vesicatoria reveal that these genes are essential for pathogenicity and assist in the survival of epiphytic bacteria [65]. These microorganisms are gram-negative and pathogenic γ-proteobacteria of many plant species of economic interest. The ncRNA MicC has 97 nucleotides, is classified as trans-encoded antisense, and is co-located with the murI and smoked genes and with the gst gene (sense). In E. coli, the MicC ncRNA is located between the ompN and ydbk genes, and regulates OmpC protein expression by pairing with the mRNA leader sequence and inhibiting ompC mRNA binding to the ribosome [47,65]. In the genome of H. seropedicae SmR1, the ompC gene is located at the Hsero_1282 locus, the ompA gene is located at the Hsero_1287 locus and the ompR gene is located at the Hsero_1518 locus. With the aid of the TargetRNA2 tool, it can be observed that the MicC ncRNA in H. seropedicae SmR1 targets the rscB gene located at the Hsero_1538 locus, which acts as a transcriptional regulator that has a receptor domain for the OmpR protein. The ncRNA MicC was expressed in all the tested conditions and achieved higher expression observed in Adhered-corn-3-days-2 and Aderido-maize-3-days-1 conditions, with a higher level of expression with regard to the other conditions. In addition, it is involved in the transcriptional expression of the rcsB gene when it is adhered to maize. In H. seropedicae SmR1, the IsrD ncRNA has 62 nucleotides, and is co-located with the mmr and Hsero_0991 genes, together with the Hsero_0992 gene (antisense sense). It has the highest expression profile in RNA-seq material under Planktonic-corn-3 days-2 and NFbHPN Malate-High-Oxygen-2 conditions. The IsrG ncRNA has 88 nucleotides, is co-located with the fimV and asd genes, and with Hs_noco_697 (sense sense), and is only expressed in the NFbHPN Malate-High Oxygen condition. In Salmonella typhimurium ncRNA, IsrG is involved in the response to cold shock and acid pH, in Int. J. Mol. Sci. 2019, 20, 46 14 of 20 the stationary phase of growth [66]. According to the literature, genes from the ncRNA-isr family have a promoter sequence superimposed on the 50 end or the 30 end of the neighboring gene [67]. In H. seropedicae SmR1, it was observed that the IsrD gene is superimposed on the 30 end of the Hsero_0991 gene, already for IsrG, expressed in the NFbHPN Malate-High Oxygen condition; it was also observed that it overlaps the fimV gene. The TargetRNA2 computational tool was used to predict ncRNA targets in H. seropedicae SmR1. This program was specifically designed for the prediction of mRNA targets of ncRNAs with activity in trans. As a result, a wide range of mRNA targets were observed, regardless of the ncRNA that was analyzed, because after transcription the cis-encoded ncRNA make short and perfect pairing with their targets and the trans-encoded ncRNAs make long and imperfect pairing [68]. The number of targets for each ncRNA was variable. For example, ncHSmR1_02 had 11 targets, ncHSmR1_33 had 45 targets, and ncHSmR1_23 with 99 targets. In the case of some ncRNAs, such as ncHSmR1_01, there was no prediction of targets. The mRNA targets predicted in H. seropedicae SmR1 triggered by different biochemical mechanisms, for example, some of the targets of ncRNA 4.5S are as follows: argB (acetylglutamate kinase), Hsero_3355 (lipoprotein), glk (glucokinase), htpX (heat shock protein HtpX), Hsero_2759 (transcriptional regulatory protein). The ncHSmR1_45 with 11 targets, ncHSmR1_149 with 45 targets, and ncHSmR1_104 with 99 targets, ncHSmR1_01 with 16 targets, ncHSmR1_140 with 5 targets. Moreover, in the case of some non-coding RNAs, such as ncHSmR1_42, there was no prediction of targets. This was probably because the mRNA sequences deposited in the TargetRNA2 tool, did not match ncHSmR1_42. The mRNA targets predicted in H. seropedicae SmR1 are related to different biochemical mechanisms, for example, among the 4.5S RNA targets are: argB (acetylglutamate kinase), Hsero_3355 (lipoprotein), glk (glucokinase), htpX and HtpX (Heat shock protein), and Hsero_2759 (transcriptional regulatory protein). These results should lead to a better understanding of the participation of this type of RNA in the regulation of the metabolism of Herbaspirillum seropedicae SmR1. Among the mRNA ratios for the predicted targets, an analysis was conducted of the likely target ncRNA-RNA interaction site, with an emphasis on those cases in which this interaction occurs near or over the RBS region. The first experimental evidence for the binding of an ncRNA to the ribosomal binding site of a messenger RNA and blocking the binding site, as a result of the base pairing between micF antisense RNA and the target mFNA mRNA, has been described in Escherichia coli in the final 50 regions of the latter [69]. Three ncRNAs were identified in H. seropedicae SmR1, with expression observed in RNA-seq material. The results indicate the existence of cis-encoded ncRNA, trans-encoded ncRNA, riboswitch, and CRISPR in H. seropedicae SmR1. Micc, IsrK, IsrD, and IsrG Hfq-dependent ncRNAs were detected among these. The number of target mRNAs predicted by the TargetRNA2 tool for the H. seropedicae SmR1 ncRNAs varied considerably and there was also a variation in the position of the mating region in relation to the AUG codon. The Infernal 1.1.1 tool did not detect ncRNAs in H lusitanum P6-12 and Herbaspirillum sp. B65.

4. Materials and Methods

4.1. Genomes of Bacteria of the Genus Herbaspirillum The genomes of H. seropedicae SmR1, H. seropedicae Z67, H. lusitanum P6-12, H. hiltneri, H. frisingense GSF30 N3, Herbaspirillum spp. strain B65, H. rubrisubalbicans M1, H. autotrophicum IAM 14942, H. huttiense subsp. putei, Herbaspirillum spp. strain B501, Herbaspirillum spp. strain GW103, Herbaspirillum spp. strain RV1423, H. rhizosphaerae UMS-37, H. rubrisubalbicans spp. strain Os34, H. rubrisubalbicans spp. strain Os45, and Herbaspirillum AU14040 were obtained from the database of the National Center for Biotechnology Information (NCBI) search. Species of Herbaspirillum, with access codes and date described, are in Supplementary Material S1. Int. J. Mol. Sci. 2019, 20, 46 15 of 20

4.2. RNA-Seq Data The researchers of the Biological Nitrogen Fixation (BNF) Centre kindly provided gene expression data of H. seropedicae SmR1. The in-plant culture and/or inoculation conditions are described in Supplementary Material S3.

4.3. Prediction of ncRNAs in Silico The H. seropedicae SmR1 genome was screened with the INFERence tools of RNA Alignment by employing a covariance model to show probabilistic proﬁles of sequences and secondary structures of RNA families [70]. The covariance model is a special case of a probabilistic mechanism that seeks to combine the consensus sequence with the secondary consensus structure of a given RNA. In many cases it is able to identify homologous RNAs that have a conserved secondary structure but low primary sequence conservation [71]. Infernal 1.1.1 available link http://eddylab.org/infernal/; accessed on 18 February 2016), consists of ﬁve programs: cmbuild, cmcalibrate, cmsearch, cmscan, and cmalign. The following command line was used: cmscan-o sequenceSMR1.out-tblout sequenceSMR1.tbl-T 24-notrunc Rfam.cm.sequenceSMR1 where sequenceSMR1, which represents the genome of the H. seropedicae SmR1 bacterium, has been replaced by the genomes listed in Section 4.1.

4.4. ncRNA Sequence Annotation The ncRNAs sequences were annotated using the Rfam online database. This database contains a collection of ncRNA families represented by sequences of manually edited alignments, consensus secondary structures, and annotations taken from taxonomic sources and ontology [70]. The base is a broad and diverse source of ncRNAs and includes 2791 families of ncRNAs, with information on various types of ncRNAs throughout the three life domains and viruses. Infernal 1.1.1 was used to make multiple sequence alignments by means of the covariance model. In addition to the annotation of ncRNAs, Rfam 14.0 classiﬁes ncRNAs and provides bibliographical references for each family, links to the PDB (Protein Data Bank), ENA (European Nucleotide Archive), and Gene Ontology (GO). The ncRNA candidates were also compared with the online Ribe dataset [72] so as to identify putative riboswitches. This database is capable of recognizing conserved sequences and known riboswitches located upstream of orthologous genes in multiple genera [73]. Also, the ncRNA candidates were compared with the NCBI database using the BlastX/N webserver.

4.5. Mapping and Visualization of Sequence Reads We masked the rRNA sequences through a cross-match program and carried out a recursive read trimming at 50 and 30 to 35 nucleotides using a Perl script to map the short reads required for targeting the genome. The resulting Mate-Paired reads were aligned to the H. seropedicae SmR1 genome using the SHRiMP alignment tool [74]. The program was set up to tolerate three mismatches. We used SAMtools available link http://samtools.sourceforge.net/; accessed on 17 February 2016) [75] to convert date into the SAM/BAM format. Mapped RNA-seq reads in BAM format were visualized in the Artemis genome browser [76].

4.6. Transcriptome Analysis The standardization of the samples sequenced on the SoliD platform was carried out by the RPKM (reads per kilobase of transcript per million mapped reads) method, which estimates the gene expression value of a gene by measuring the density of the readings in a gene region of interest, and standardizing the readings counts in their exonic regions, compared with the original size of the gene or exon [77]. Int. J. Mol. Sci. 2019, 20, 46 16 of 20

4.7. Target Prediction The TargetRNA2 prediction tool was employed to target mRNAs that base-base with ncRNAs [68,78]. It calculates the hybridization score and the statistical signiﬁcance of interactions between mRNA-ncRNA [79]. The tool uses a wide range of features to identify the target mRNAs, among them the conservation of ncRNA. This feature makes a comparison of the available sequence in GenBank in terms of the deposited genome (replicon) and indication of regions that have a greater conservation of sequences that are prone to undergoing mRNA/cRNA interactions. Another feature is the accessibility structure of the mRNA mRNA/ncRNA, which is weighted by its stability and regions of interactions. In addition, account is taken of the energy hybridization regions of the mRNAs with a low energy index for hybridization in one or more regions of interactions with potential targets [68].

4.8. Identifying CRISPR A computational tool to identify CRISPR classiﬁcation repeat conservation was used. This allows the independent grouping and determination of conserved sequence families, structural motifs suitable for endoribonucleases, and evolutionary relations [80]. CRISPRFinder is an interface that provides a detailed analysis of CRISPR genomic sequences [81].

5. Conclusions The method used by Inferna1.1.1. for prediction of ncRNAs in the genus Herbaspirillum spp. prioritizes the identity of the various classes of ncRNAs. This RNA alignment inference has perspective combining the biological premise on associations of DNA sequences to RNA structure and sequence similarities. At the same time incorporation of the covariance models (CMs) scores a combination of sequence consensus and secondary RNA structure consensus, therefore, ncRNAs in the genus Herbaspirillum spp. Were identiﬁed, given the importance of ncRNA expression in seq RNA in in the strain Herbaspirillum seropedicae SmR1. The results provide a better understanding of the mechanism of post-transcriptional regulation of ncRNAs due to the interaction of mRNAs mRNAs and the diversity of ncRNAs classiﬁcation in the genus Herbaspirillum spp.

Supplementary Materials: Supplementary materials can be found at http://www.mdpi.com/1422-0067/20/1/ 46/s1. Author Contributions: A.C.G. predicted of ncRNAs in the genus Herbapirillum spp. and wrote the manuscript. V.L.P.d.S., T.C.S.C., L.M.C. and H.G. contributed to the funding acquisition and revision of the manuscript. Funding: This study was financed in part by the Coordination of Improvement of Higher Education Personnel—Brazil (CAPES)—Finance Code 001. Conflicts of Interest: The authors declare no conflict of interest.

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