Mol Biol Rep DOI 10.1007/s11033-014-3411-3

Genome organization in Mycoplasma hyopneumoniae: identification of promoter-like sequences Franciele Maboni Siqueira • Shana de Souto Weber Amanda Malvessi Cattani • Irene Silveira Schrank

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Received: 30 June 2013 / Accepted: 11 May 2014 Ó Springer Science+Business Media Dordrecht 2014

Abstract Information related to open reading frame (ORF) organization, transcription regulation and promoter sequence has been available for the Mycoplasma hyopneumoniae 7448 genome, demonstrating that the ORFs are continuously transcribed (cotranscription) in large clusters. A species-specific position-specific scoring matrix was applied to scan for putative promoters upstream of all coding sequences on a genome scale in M. hyopneumoniae. This study consisted of a detailed in silico promoter localization analysis by scanning the position-specific promoters upstream of predicted ORF clusters (OCs) and mCs (monocistronic genes) in the M. hyopneumoniae whole genome; this was combined with experimental data for the promoterless ORFs. Promoter-like sequences were found in 86 % of the OCs (from the OC first gene) and in

85 % of the mCs. A transcription analysis of the promoterless ORF was performed by RT-PCR. This strategy allowed the definition of a specific promoter sequence for all OCs and mCs indicating that all the transcriptional units are preceded by putative promoter sequences (matrix and manually located) and providing evidence for functional gene organization in the M. hyopneumoniae genome. These results shown that the species-specific, position-specific scoring matrix for promoter prediction is effective, further increasing the knowledge of gene organization and transcription initiation in mycoplasmas. Keywords Transcriptional units
Species-specific matrix promoter
Transcription initiation
Genome organization

Introduction Electronic supplementary material The online version of this article (doi:10.1007/s11033-014-3411-3) contains supplementary material, which is available to authorized users. F. M. Siqueira (&)
S. de Souto Weber
A. M. Cattani
I. S. Schrank Centro de Biotecnologia, Universidade Federal do Rio Grande do Sul (UFRGS), Porto Alegre, Brazil e-mail: [email protected] I. S. Schrank e-mail: [email protected] F. M. Siqueira Programa de Po´s-Graduac¸a˜o em Cieˆncias Biolo´gicasBioquı´mica, UFRGS, Porto Alegre, Brazil I. S. Schrank Departamento de Biologia Molecular e Biotecnologia, Instituto de Biocieˆncias, UFRGS, Porto Alegre, Brazil

Mycoplasma hyopneumoniae is a porcine bacterial pathogen and is the leading cause of enzootic pneumonia [1]. Similar to other mycoplasmas, this bacterium has no cell wall and a reduced genome with a limited biosynthetic metabolism. The availability of an increasing number of completely sequenced mycoplasmal genomes, including four M. hyopneumoniae strains, makes it possible to explore gene organization in this genus [2–4]. Recently, information related to Open Reading Frame (ORF) cluster (OC) organization, transcription regulation and promoter sequences has been available for the M. hyopneumoniae 7448 genome [5, 6]. The global assessment of OC organization of the M. hyopneumoniae genome by both in silico and in vitro approaches demonstrated that the ORFs are continuously transcribed (cotranscription) in a large cluster (OC); every

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gene is transcribed in the same direction with no intervening gene transcribed in the opposite one [5]. These authors had performed a systematic analysis of the conservation of gene order organization and synteny among three M. hyopneumoniae genomes; two pathogenic strains (7448, 232) and one non-pathogenic strain (J). The fact that the ORFs are organized into clusters is a feature of the genome of M. hyopneumoniae 7448, and it is shared with other pathogenic and non-pathogenic strains [5] and also with other mycoplasmas [7]. Although several mycoplasma species have had their genome sequenced, very little information related to promoter sequence and transcription mechanisms is available for these organisms. Similar to other mycoplasmas, M. hyopneumoniae has only one sigma factor, few regulatory proteins and no transcription termination Rho factor [3]. Previous studies, using an E. coli sigma-70 promoter matrix were unsuccessful in detecting promoters in mycoplasma genomes [8, 9]. Therefore, to identify RNA polymerase binding regions (putative promoters) on a genomic scale in M. hyopneumoniae a species-specific position-specific scoring matrix (PSSM) was built and used to scan for putative promoters upstream of all coding sequences [6]. As expected, a pattern similar to a sigma-70 -10-element was found but no -35 promoter element was identified in association with 169 ORFs. Analysis of the M. hyopneumoniae genome revealed that the above promoter-like sequences were found upstream of only 26 % of the total ORFs, indicating that some putative promoters were not detected. The limited number of ORFs with putative promoter signals is probably related to the criteria for promoter prediction applied in this study that had used the higher threshold score of 6.5 as the cut-off value [6]. However, up till now, this is the first wholegenome analysis that was able to identify putative promoter regions in M. hyopneumoniae. To better understand gene organization and transcription in M. hyopneumoniae, we have performed a detailed in silico promoter localization analysis combined with experimental data for the promoterless ORFs. Moreover, we have also included the lowest score of 4.2 found in the experimentally defined promoters [6] for scanning the position-specific promoters upstream the predicted OCs and monocistronic genes (mC) in the M. hyopneumoniae whole genome. This strategy allowed the definition of a specific promoter sequence for each OC or mC indicating that the majority of the genes are arranged in transcriptional units. However, a subset of the OCs could be independently transcribed due to the presence of internal promoters. This makes the species-specific position-specific scoring matrix for promoter predictions possible, further increasing the knowledge of transcription initiation in mycoplasmas.

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Fig. 1 Experimental design workflow correlating genome organization with promoter location in M. hyopneumoniae 7448

Materials and methods In silico analysis of predicted promoters upstream of ORF clusters and monocistronic genes The optimum matrix parameters defined by Weber et al. [6] were used to scan the upstream sequences of all 657 M. hyopneumoniae ORFs (MHP7448_GenBank: NC_007332) for the presence of putative promoters using a threshold score of 6.5 (named P6.5 promoter) and also the threshold between scores 6.5 and 4.2 (named P4.2 promoter), the smallest weight score obtained for the experimentally defined promoters. The position of promoter-like sequences upstream of each mC and the first gene of each M. hyopneumoniae OC, previously mapped by Siqueira et al. [5] (genome reference: MHP7448_GenBank: NC_007332), was manually located and analyzed using the Artemis Release 10.5.2 software [10]. Each putative promoter sequence position was evaluated in the M. hyopneumoniae genome considering upstream regions up to 350 base pairs from the start codon of the mCs and also of the first transcribed gene in the OC. Moreover, regions upstream of ORFs predicted to be inside ORF clusters were considered internal promoter-like sequences, and putative promoters in the incorrect orientation related to the start codon were excluded. Experimental characterization of the promoterless OCs’ and mCs’ transcription units was performed by reverse transcriptase PCR (RT-PCR). Furthermore, some transcription units predicted in the M. hyopneumoniae 7448 strain with internal promoters were characterized by quantitative RT-PCR. A final global analysis was performed taking into consideration the promoter position together with the localization of the internal promoters and

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the experimentally analyzed transcription units. Figure 1 shows the schematic representation of the search procedure used to establish the correlation between promoter localization and gene organization in the M. hyopneumoniae genome. Bacterial strain and culture conditions The M. hyopneumoniae strain 7448 was cultured in a 25 ml Friis broth [11] at 37 °C for 24 h with gentle agitation in a roller drum. Total RNA was isolated with RNeasy Mini Kit (Qiagen, USA). For cell lysis, 0.7 ml of RNeasy Lysis Buffer (RLT buffer) in the presence of 0.134 M b-mercaptoethanol was used per cultivation flask. The purification was done according to the manufacturer’s instructions, with on-column DNaseI digestion using the RNase-Free DNase Set (Qiagen, Germany) and a second round of treatment with DNase I (Fermentas, USA). The DNA absence was controlled to below PCR-detectable levels. The extracted RNA was analyzed by gel electrophoresis and quantified in the QubitTM system (Invitrogen, USA). Reverse transcriptase PCR Reverse transcription was performed with M-MLV RT (Moloney Murine Leukemia Virus Reverse Transcriptase—Invitrogen, USA) with 1 lg of RNA and pd(N)6 random hexamer (GE Healthcare, USA). Specific RT-PCR primers (Supplementary Table S1) were designed based on the M. hyopneumoniae 7448 genome sequence to amplify the coding regions of predicted annotated sequences on the GenBank database (NC_007332). Gene-specific primers were designed using Vector NTI Advance 10 (Invitrogen, USA). Reactions were performed with Go-Taq Polymerase (5U–Promega, USA) with the following cycling parameters: 94 °C for 2 min, then 30 cycles of 94 °C for 30 s, melting temperature (Tm) for 30 s (Tms were indicated in Supplementary Table S1), 72 °C for 1 min, and a final step at 72 °C for 10 min. For each primer pair a set of three control reactions was performed: (1) a reverse transcription reaction control (without RT enzyme), (2) a PCR positive reaction control (with genomic DNA of M. hyopneumoniae as template) and (3) a PCR negative reaction control (no template DNA). Reaction products were analyzed in 1 % agarose gels and fragments of the expected size were precipitated with 1 lg of tRNA (Invitrogen, USA) and 2.5 V of absolute ethanol. The pellets were resuspended in 15 ll of ultrapure water and sequenced in the MegaBACE 1,000 DNA Analysis System automated sequencer (GE HealthCare, USA). Sequences quality was evaluated by Phred20 score.

Quantitative RT-PCR Quantitative RT-PCR (qPCR) was utilized to confirm the presence of internal promoters. Total RNA (1 lg) was reversed transcribed into cDNA with M-MLV RT (Invitrogen, USA) according to the manufacturer’s protocols using pd(N)6 random hexamer (GE Healthcare, USA). Specific qPCR primers were designed (based on the M. hyopneumoniae 7448 genome sequence-GenBank: NC_007332) to target ORFs within selected OCs with internal promoters (Supplementary Table S1). Quantitative RT-PCR was performed using 1:50 cDNA as template and Platinum SYBR Green qPCR SuperMix-UDG (Invitrogen, USA) on the StepOne Real-Time PCR System (Applied Biosystems, USA). MHP7448_0333 ORF was used as control for all the experiments [12, 13]. Gene-specific primers were designed using Vector NTI Advance 10 (Invitrogen, USA). A dissociation curve was carried out for each primer pair showing only one melting temperature. The qPCR reactions were carried out at 90 °C for 2 min and 95 °C for 10 min followed by 40 cycles of 95 °C for 15 s and 60 °C for 1 min each. The relative gene expression was calculated using the comparative CT method [14]. The threshold cycles (CT) of each test target represents the average of three reactions and the values were normalized according to the CT of the control product. For each target ORF, four independent biological replicates were performed. Expression data was normalized by subtracting the mean reference gene CT value from individual CT values of corresponding target genes (DCT). One-way ANOVA followed by Dunnett’s multiple comparison test or a onetailed unpaired t test were used to determine if the relative expression values of the genes within an OC were significantly different. Statistical analyses were performed using GraphPad Prism 6 software (GraphPad Software), considering a 5 % significance level.

Results and discussion Screening for promoter like sequences upstream OCs and mCs Aiming to contribute to the understanding of gene clustering in the organization of the mycoplasma chromosome, a promoter localization analysis was performed on the M. hyopneumoniae whole genome. To investigate the presence and localization of promoter-like sequences associated with ORFs in M. hyopneumoniae, a systematic analysis of all upstream regions of the 116 OCs and 33 mCs was performed.

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Fig. 2 Number of OCs and mCs with promoter-like sequences by position-specific scoring matrix screening, a promoter-like sequence localized within 350 bp from the mCs start codon and from the start codon of the first ORF of each OC. P6.5 promoter: OCs and mCs containing putative promoter sequences defined by the threshold score 6.5 and above; and P4.2 promoter: OCs and mCs with putative promoter sequences defined by thresholds between scores 6.5 and 4.2. b Distribution of internal promoters within ORF clusters. Asterisks Total number of OCs and mCs in M. hyopneumoniae 7448 genome

Promoter sequence prediction by Weber et al. [6] was based in the sequence that comprised up to 250 bases upstream and 50 bases downstream of the start codon from M. hyopneumoniae genes. Based on these previously information we have used two criteria for accept a sequence as promoter. The first was the sequence localized at a maximum distance of 350 bases upstream of the start codon of each mC and upstream of the start codon of the first gene of each OC. The second was the presence of promoters sequences with a threshold score greater than 4.2 defined by the species-specific position-specific scoring matrix [6]. Using these criteria, promoter-like sequences were identified in 86 % of OCs (100 of 116 OCs) and 85 % of mCs (28 of 33 mCs) in the M. hyopneumoniae genome (Fig. 2a; Supplementary Table S2). The distance of the promoters from the start codons varied between -13 to -348 bases, although the majorities were located approximately at -1 to -25 bases from the predicted start codon (Fig. 3a). Our results are in agreement with previous work that had performed the speciesspecific position-specific scoring matrix analysis taking into consideration the 250 bases of noncoding sequences upstream of the start codon of the whole M. hyopneumoniae genome and found that the majority of the promoter

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Fig. 3 Distance of the promoter site to the ORFs’ start codon of the M. hyopneumoniae genome. a Represents the distance (in bases) of the promoter site from the mCs start codon and from the start codon of the OCs first ORF, and b represents the distance of the internal promoter site located upstream of each ORF

sequences were located between 1 to 100 bases upstream of the start codon [6]. However, heterogeneous promoter localization has been found in M. hyopneumoniae [6] and in M. pneumoniae [8] genomes with some of them located around -220 bases from the predicted start codon. In some ORFs, such as OC06 and OC99, putative promoters were localized at 348 bases and 246 bases, respectively, upstream of the start codon (Supplementary Table S2). Although no transcriptional start sites were mapped for OC06 and OC99, experimental data revealed the presence of transcripts for these ORF clusters (data not shown). Number variation of promoter sequences among OCs and mCs In 63 OCs (promoter position upstream the start codon from the OC first gene) and 16 mCs, a single promoter was located representing 54 and 48 %, respectively, of total OCs and mCs in the M. hyopneumoniae genome. However, the presence of additional promoter sites was identified in 12 mCs and in 37 OCs (Supplementary Table S2).

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Interestingly, 23 of the 37 OCs (62 %) and five of the 12 mCs (42 %) revealed the presence of additional putative promoter sites defined as a P6.5 promoter together with the P4.2 promoter (Fig. 2a). Moreover, the localization of the additional promoter sites was variable relative to the start codon of the mC or of the start codon of the first gene of the OC (Supplementary Table S2). The presence of these additional promoter sites could be related to multiple transcriptional start sites suggesting that M. hyopneumoniae promoters can be regulated by different signals or factors. Previous studies have also demonstrated the presence of multiple promoter sites in the Caulobacter crescentus genome and have identified motifs for activators or repressors that could be related to transcription regulation [15]. Differences in promoter number and position have been found among OCs and mCs in the M. hyopneumoniae genome. Analysis of mCs and OCs containing coding sequences for essential bacterial enzymes, for example, revealed the presence of single promoter sites in OC01, OC06, OC08, and OC36 containing ORFs encoding chromosomal replication initiation protein, recombinase A, molecular chaperone DnaK and tRNA synthetases, respectively, among others, and multiple promoter sites in OC04 (with ORFs coding for DNA topoisomerase IV subunits), OC28 (30S/50S ribosomal protein cluster), OC70 (with ORF coding for the elongation factor P), mC14 (ORF coding for transcriptional regulator) and mC24 (ORF coding for DNA gyrase subunit A), among others. Interestingly, OC04 has two P4.2 promoter sites, mC14 and mC24 have two P6.5 promoter sites and in OC28 and OC70 one P4.2 promoter and one P6.5 promoter have been localized upstream of each OC first ORF. Promoter determined by the threshold score between 6.5 and 4.2 are true promoters The presence of a P4.2 promoter site, determined by the threshold score between 6.5 and 4.2, has been previously estimated to be false-positive [6]. However, a P4.2 promoter site was localized in 14 mCs and upstream of the first ORF in 55 OCs (Fig. 2a). Moreover, only a P4.2 promoter type was localized in approximately 32 % of OCs (32 P4.2 promoter) and mCs (nine P4.2 promoter) in the M. hyopneumoniae genome, and previous studies [5] had performed a transcript experimental analysis of OC55, OC98, OC109 and OC111 showing that ORFs with only a P4.2 promoter are transcribed (Supplementary Table S2). The P4.2 promoter was also located in experimentally characterized transcriptional start sites in selected genes including licA (OC07), P97 ORF (OC29), pyrH (OC90), uvrC (mC03), ktrA (mC25) and dam (mC31), on the same genome strain used in this present study, the M. hyopneumoniae 7448 [6]. The Supplementary Table S3 presents the promoters location (P4.2 promoter) of

these six genes whose TSSs have been identified. In conclusion, taking into consideration the experimentally demonstrated transcript OCs (that have promoter sequence with only the threshold score between 6.5 and 4.2, named promoter P4.2), and the frequency of P4.2 promoter in whole M. hyopneumoniae genome, we believed that is possible to suggest that some P4.2 promoters, previously considered false-positive by Weber et al. [6] are true promoters. Promoterless ORFs display putative TATA boxes and are transcribed Promoter scanning of the M. hyopneumoniae genome for both P6.5 and P4.2 promoters revealed that putative signals were not found on five mCs (15 %) and 16 OCs (14 %) (Fig. 2a and Supplementary Table S4). In order to analyze the transcription of the promoterless mCs and OCs, RTPCR was performed. Specific primers were designed to amplify the five mCs and the first ORF of the 16 OCs; the genome localization of the relevant primer-pairs are shown in Fig. 4a. Amplification products for all target ORFs were analyzed by DNA sequencing revealing the presence of transcripts from the five mCs and from the 16 OCs first ORF (Supplementary Tables S2, S4). To better understand the promoterless ORFs, the region upstream of the start codon of each ORF was manually analyzed and putative TATA boxes were located (Supplementary Table S5). Detailed analysis of the 21 ORFs’ regulatory regions shows the presence of TATA boxes located 7–283 nucleotides upstream of the start codon. However, the presence of the semi-conserved -16 element found by Weber et al. [6] was not localized in the regulatory regions of the 16 OCs and five mCs (Supplementary Table S5). Previous results had demonstrated that among the 23 promoters experimentally mapped, the pattern detected by the matrix shows the P6.5 promoter in 69.5 %, the P4.2 promoter in 26 % and only the recA promoter did not score. The patterns TATAAT or TAAAAT were identified in 21 genes, and in recA (AAAAAT) and ktrA (TACAAT) two other patterns were characterized [6]. Among the 21 promoterless ORFs analyzed, the canonical sigma 70 promoter TATAAT was located in the upstream region of mC02 and upstream the first ORF in OC21, OC45, OC56, OC101 and OC106 (Supplementary Table S5). The patterns most frequently found were TAAAAT and AAAAAT located in the upstream region of four mCs and upstream the first ORF of 10 OCs. Only in the upstream sequence of OC20 were those motifs not localized, although the presence of a TTAATT pattern was located 93 nucleotides from the start codon (Supplementary Table S5). These findings suggest that the matrix predictive performance was unable to locate promoters in regulatory regions lacking the -16 element and emphasizes the existence of

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Fig. 4 Schematic diagram displaying the putative promoter sites in a region of M. hyopneumoniae 7448 genome. a OC89 representation: primers used for OC89 internal promoter sites, qPCR analyses are represented by horizontal black arrows below genes. b OC91 represents a promoterless OC: horizontal black arrows below genes represent the location of primer pairs used in RT-PCR analyses, White

arrowheads indicate the direction of transcription and grey lines represent the intergenic regions. The vertical arrows indicate the predicted promoter sites by position-specific scoring matrix in the appropriate orientation (Open arrows: P4.2 promoters; closed arrows: P6.5 promoters). Visualization by the software Artemis

transcriptional regulatory elements in the upstream region of these OCs or mCs.

internal promoters were characterized and also variations in the number of internal promoters in the upstream region of each ORF. The OC89 is a typical example of an OC containing differences in the number of internal promoters (Fig. 4b). In this OC, one putative P6.5 promoter site was localized between deoC and upp and between the MHP7448_0525 and lon genes, while three P6.5 promoter sites and two P4.2 promoter sites were located between the lon and tuf genes. The effect of internal promoters could be related to the production of different transcripts, thereby adding plasticity to OC organization in the M. hyopneumoniae genome. In order to establish the role of the internal promoters in M. hyopneumoniae cells, the transcriptional expression level was evaluated in some OCs containing internal promoters. Within the OCs with internal promoters, we have searched for protein functions identified by ontology; OC10, OC89 and OC60 (as control) were selected for qPCR analysis and the transcription quantification was performed for all ORFs from each OC. Supplementary Table S2 shows the distribution of internal promoters sites in these OCs, and a schematic representation of internal promoter sites together with qPCR primers is presented in Fig. 4b. OC10 composed of two genes coding for an amino acid permease and NADH oxidase (MHP7448_0081 and nox,

ORFs can be transcript independently by alternative internal promoters In the M. hyopneumoniae genome, ORFs are transcribed together in long transcriptional units [5]. However, putative promoter sequences have been located in the upstream regions of internal ORFs in many ORF clusters [6], and independent transcription has been demonstrated from intergenic regions and between internal regions within ORF clusters giving rise to transcripts of different lengths [16]. Therefore, the M. hyopneumoniae genome was scanned for the presence of promoter-like sequences, and global analyses demonstrated the presence of internal promoters in 82 ORF clusters representing 70 % of all OCs (Fig. 2b). Similarly to distance of the promoter site from the mCs start codon and from the start codon of the OCs first ORF, the distance of the internal promoter sites upstream of ORF’s were at 1–25 bases from the predicted start codon (Fig. 3b). The presence of the P6.5 promoter was detected as an internal promoter in the majority of the OCs, and in 15 OCs an equal distribution of the P6.5 and P4.2 promoters was identified. Among the 82 OCs, differences in the number of

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Fig. 5 Transcriptional expression level of M. hyopneumoniae 7448 OCs with internal promoters, qRT-PCR was used to measure the transcription expression of all genes in OC10, OC60, OC89. OC60 represents an ORF cluster without an internal promoter and was used as a control. The bars represent the genes relative expression

normalized to MHP7448_0333 expression in the corresponding samples. Statistical analysis considering a 5 % significance level demonstrated differential expression between MHP7448_0081 and nox, between deoC and upp, between MHP7448_0525 and lon, and between lon and tuf

respectively) and OC89 composed of five genes coding for deoxyribose-phosphate aldolase, uracil phosphoribosyltransferase, hypothetical protein, heat shock ATP-dependent protease and elongation factor Tu (deoC, upp, MHP7448_0525, lon and tuf, respectively) are typical representatives of M. hyopneumoniae gene organization. Previous studies have demonstrated that, in this bacterium, 85 % of the ORF clusters contain a number of two to eight ORFs with variation in the functional categories of the encoded products [5]. Functional validation of the internal promoters located in OC10 and OC89 was obtained by the differential expression level detected for the nox gene in OC10, and upp, lon and tuf genes in OC89 (Fig. 5). Interestingly, the tuf gene is highly expressed in the tested conditions, hence pointing to a possible role of the five internal promoters in differential transcription in M. hyopneumoniae. These results suggest that some internal promoters are functional in the M. hyopneumoniae genome, thus, representing alternative sites of transcription initiation. Previous reports have also suggested that a subset of the OCs could be independently transcribed due to the presence of internal promoters correlating with a possible complex transcriptional organization in M. hyopneumoniae genomes [5, 6]. Analyses of the M. pneumoniae transcriptome have also demonstrated the presence of polycistronic operons with alternative transcripts [7]. Moreover, more than one promoter site has been located in some operons in Bacillus subtilis [17]. These data are in agreement with the results determined for M. hyopneumoniae and are indicative that the effect of internal promoters would result in the production of different transcripts. The presence of internal promoter sites localized in 70 % of M. hyopneumoniae ORF clusters suggests an alternative mechanism for differential transcription in this bacterium. However, the presence of a promoter site

located exclusively in the upstream region of the first ORF was detected in 34 OCs (Supplementary Table S2; Fig. 2b). Detailed analysis of the 34 OCs revealed variation in ORF number, length and also in the functional categories of the encoded products. Interestingly, some of these OCs contain ORFs encoding products important for essential metabolic processes, such as OC04 (DNA topoisomerase IV subunit A and B) and OC99 (DNA polymerase III- DnaE and translation initiation factor IF-2). Taken together, these results contribute to increase our knowledge of gene organization and transcription in the M. hyopneumoniae genome.

Conclusion This is the first work on M. hyopneumoniae that combine whole genome analysis on gene organization and promoter localization. The M. hyopneumoniae transcription unit architecture is characterized by the presence of long transcriptional units containing ORFs that are highly variable in the functional categories of the encoded products. We were able to demonstrate that all the transcriptional units and monocistronic ORFs are preceded by putative promoter sequences (PSSM and manually located) providing evidence for functional gene organization in the M. hyopneumoniae genome. Although ORF clustering is important for the organization of the M. hyopneumoniae genome, the presence of alternative internal promoters suggests that a subset of ORFs within the ORF clusters could be independently transcribed. Promoter motifs are difficult to establish for AT rich genomes and in this paper were able to validate the species-specific position-specific scoring matrix previously defined by Weber et al. [6]. Therefore, the knowledge generated on the dynamics of genome organization and on the regulatory elements should

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contribute to understanding unexplored mechanisms of mycoplasma transcriptional regulation. Acknowledgments This work was supported by grants from the Brazilian National Research Council (CNPq).

References 1. Ross RF (1992) Mycoplasmal disease. In: Leman AD, Straw BE, Mengeling WL, D’Allaire S, Taylor DJ (eds) Diseases of swine. Iowa State University Press, Ames, p 537-351 2. Minion FC, Lefkowitz EL, Madsen ML, Cleary BJ, Swartzell SM, Mahairas GG (2004) The genome sequence of strain 232, the agent of swine mycoplasmosis. J Bacteriol 186:7123–7133 3. Vasconcelos AT, Ferreira HB, Bizarro CV et al (2005) Swine and poultry pathogens: the complete genome sequences of two strains of Mycoplasma hyopneumoniae and a strain of Mycoplasma synoviae. J Bacteriol 187:5568–5577 4. Liu W, Feng Z, Fang L, Zhou Z, Li Q, Li S, Luo R, Wang L, Chen H et al (2011) Complete genome sequence of Mycoplasma hyopneumoniae strain 168. J Bacteriol 193:1016–1017 5. Siqueira FM, Schrank A, Schrank IS (2011) Mycoplasma hyopneumoniae transcription unit organization: genome survey and prediction. DNA Res 18:413–422 6. Weber SS, Sant’anna FH, Schrank IS (2012) Unveiling Mycoplasma hyopneumoniae promoters: sequence definition and genomic distribution. DNA Res 19:103–115 7. Gu¨ell M, Noort V, Yus E, Chen WH, Leigh-Bell J, Michalodimitrakis K, Yamada T, Arumugam M, Doerks T et al (2009) Transcriptome complexity in a genome-reduced bacterium. Science 326:1268–1271

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8. Weiner JIII, Herrmann R, Browning GF (2000) Transcription in Mycoplasma pneumonia. Nucl Acids Res 28:4488–4496 9. Sinoquet C, Demey S, Braun F (2008) Large-scale computational and statistical analyses of high transcription potentialities in 32 prokaryotic genomes. Nucl Acids Res 36:3332–3340 10. Rutherford K, Parkhill J, Crook J, Horsnell T, Rice P, Rajandream MA, Barrell B (2000) Artemis: sequence visualization and annotation. Bioinformatics 16:944–945 11. Friis NF (1975) Some recommendations concerning primary isolation of Mycoplasma suipneumoniae and Mycoplasma flocculare a survey. Nordisk Veterinaer Med 27:337–339 12. Madsen ML, Puttamreddy S, Thacker EL, Carruthers MD, Minion FC (2008) Transcriptome changes in Mycoplasma hyopneumoniae during infection. Infect Immun 76:658–663 13. Moitinho-Silva L, Heineck BL, Reolon LA, Paes JA, Klein CS, Rebelatto R, Schrank IS, Zaha A, Ferreira HB (2011) Mycoplasma hyopneumoniae type I signal peptidase: expression and evaluation of its diagnostic potential. Vet Microbiol 154:282–291 14. Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2(delta delta C(T)) method. Methods 25:402–408 15. McGrath PT, Lee H, Zhang L, Iniesta AA, Hottes AK, Tan MH, Hillson NJ, Hu P, Shapiro L, McAdams H (2007) Highthroughput identification of transcription start sites conserved promoter motifs and predicted regulons. Nat Biotech 25:584–592 16. Gardner SW, Minion FC (2010) Detection and quantification of intergenic transcription in Mycoplasma hyopneumoniae. Microbiology 156:2305–2315 17. Makita Y, Nakao M, Ogasawara N, Nakai K (2004) DBTBS: database of transcriptional regulation in Bacillus subtilis and its contribution to comparative genomics. Nucl Acids Res 32:D75– D77