Gene 508 (2012) 221–228

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Short Communication

Under-representation of intrinsic terminators across bacterial genomic islands: Rhoas a principal regulator of xenogenic DNA expression

Anirban Mitra a, Valakunja Nagaraja a,b,⁎a Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore, Indiab Jawaharlal Nehru Centre for Advanced Scientific Research, Bangalore, India

Abbreviations: GI, genomic islands; IT, intrinsic termtransfer; RNAP, RNA polymerase.⁎ Corresponding author at: Department of Microbiolog

tute of Science, Bangalore-560012, India. Tel.: +91 80 229E-mail address: vraj@mcbl.iisc.ernet.in (V. Nagaraja)

0378-1119/$ – see front matter © 2012 Elsevier B.V. Allhttp://dx.doi.org/10.1016/j.gene.2012.07.064

a b s t r a c t

a r t i c l e i n f o

Article history:Accepted 30 July 2012Available online 4 August 2012

Keywords:Horizontal gene transferIntrinsic terminationHairpinRNA polymerasePathogenicity

Two transcription termination mechanisms – intrinsic and Rho-dependent – have evolved in bacteria. TheRho factor occurs in most bacterial lineages, and has been hypothesized to play a global regulatory role.Genome-wide studies using microarray, 2D-gel electrophoresis and ChIP-chip provided evidence that Rhoserves to silence transcription from horizontally acquired genes and prophages in Escherichia coli K-12, impli-cating the factor to be a part of the “cellular immune mechanism” protecting against deleterious phages andaberrant gene expression from acquired xenogenic DNA. We have investigated this model by adopting an al-ternate in silico approach and have extended the study to other species. Our analysis shows that several ge-nomic islands across diverse phyla have under-representation of intrinsic terminators, similar to thatexperimentally observed in E. coli K-12. This implies that Rho-dependent termination is the predominantprocess operational in these islands and that silencing of foreign DNA is a conserved function of Rho. Fromthe present analysis, it is evident that horizontally acquired islands have lost intrinsic terminators to facilitateRho-dependent termination. These results underscore the importance of Rho as a conserved, genome-widesentinel that regulates potentially toxic xenogenic DNA.

© 2012 Elsevier B.V. All rights reserved.

1. Introduction

Transcription involves synthesis of RNA by RNA polymerase (RNAP)on a DNA template and is functionally divided into initiation, elongationand termination (von Hippel, 1998). The last step i.e. terminationinvolves stopping of elongation, release of the RNA and dissociationof the RNAP machinery (Richardson and Greenblatt, 1996). Inbacteria, termination functions by two mechanisms — intrinsic andfactor-dependent (Peters et al., 2011; Santangelo and Artsimovitch,2011). At intrinsic terminators (ITs), termination is effected by the se-quence and structural features of the hairpin and the U-trail of the na-scent RNA (Epshtein et al., 2007). In contrast, factor-dependenttermination predominantly involves the Rho protein which shows littlepreference for any specific sequence or structure on the RNA and thetemplate DNA for its activity (Ciampi, 2006; Richardson, 2002). Rhoseems to be the major termination factor for genes that do not havean IT downstream. It has been speculated that several genes are likelytargets for Rho in vivo, although only few have been characterized(Ciampi, 2006). Historically, termination has received relatively lesserattention than the first two steps of transcription. In the recent

inator; HGT, horizontal gene

y and Cell Biology, Indian Insti-32598; fax: +91 80 23600668..

rights reserved.

post-genomics era its regulatory importance in the context of thewhole cell is being understood (Cardinale et al., 2008; Peters et al.,2009).

An outcome of the large-scale sequencing and annotation ofgenomes is the “pangenome” concept. It is now understood that hori-zontal gene transfer has played a pivotal role in the evolution of pro-karyotes (Boto, 2010; Boyd et al., 2009; Juhas et al., 2009; Ochman etal., 2000). In a nutshell, horizontal gene transfer (HGT) is the acquisitionof DNA from the environment and its integration into the genomeof therecipient species. The genes would be inherited by the daughter cells,even though they were not transmitted “vertically”. Such genes orgene clusters (henceforth, generically referred to as genomic islands(GI)), code for various protein(s) with myriad functions. Their acquisi-tion can result in “quantum leaps” by bacterial genomes (Boto, 2010;Nakamura et al., 2004). However, un-concerted expression of anyrecently-acquired gene(s) or expression of toxic proteins frombacterio-phages (Canchaya et al., 2004) (Casjens, 2003) can have disastrous ef-fects on cellular homeostasis of the host. Hence, after entering agenome,most GIs are repressed by silencingmechanisms that act at dif-ferent stages of the gene expression process (Navarre et al., 2007).

Althoughmechanisms that control initiation and repression of tran-scription in GIs have been studied, the importance of transcription ter-mination at genomic islands was noticed only recently. In Escherichiacoli, the global regulatory role of Rho has emerged from two studiesusing either microarray or ChIP-chip approaches, (Cardinale et al.,2008; Peters et al., 2009). Such studies have unambiguously shown

222 A. Mitra, V. Nagaraja / Gene 508 (2012) 221–228

that in E. coli Rho-dependent termination is important in suppressingaberrant expression from the genomic islands including prophages.

In this manuscript, we propose that the suppression of transcriptionin GIs could be a universally conserved function of Rho. We show thatRho-dependent termination indeed seems to play a similar role in reg-ulating expression at GIs across diverse bacterial phyla. Furthermore,based on the experimental understanding about the mechanism of in-teractions of Rho with the nascent RNA and RNAP (Dutta et al., 2008),we suggest that the lesser density of ITs in GI could actually facilitateRho-dependent termination.

2. Materials and methods

The program GeSTer can recognize both canonical and non-canonicalintrinsic terminators. The mode of action of GeSTer has been describedearlier (Mitra et al., 2009, 2010; Unniraman et al., 2002). All genome se-quences were downloaded in their GenBank format from NCBI (ftp://ftp.ncbi.nih.gov/genbank/genomes/Bacteria/). Sequence informa-tion about GIs was obtained from available literature, and from theNCBI lists for individual genomes. Once GeSTer has identified allthe ITs for a genome, we analyzed the intrinsic terminator-contentin the GIs of that genome. For a given GI, the density of ITs (DIT)was calculated as the [(number of ITs identified) /(number ofgenes)]×100. Similarly, the genomic DIT=[(number of ITs identifiedin genome)/(number of genes in genome)]×100.

To ascertain which transcription units (multigenic operon orsingle-gene) had an IT downstream, the gene at the 3′ end of amultigenic operon was identified from the DOOR database, and theGeSTer results for that genome were analyzed to see if that 3′ termi-nal gene had an IT after its stop codon. The HGT (IT/TU)% was calcu-lated as (number of ITs in the GI)/(number of transcription units inthe GI). The total number of transcription units in the genome wascalculated from the genome-specific statistics available at the DOORsite (http://csbl1.bmb.uga.edu/OperonDB_10142009/displayspecies.php).

Genomic (IT/TU)% is calculated as (number of ITs in the genome)/(number of transcription units in the genome).

3. Results and discussion

3.1. Rationale for the experimental design

A salient result of the microarray studies in E. coli K-12 is that,when Rho action was inhibited by the antibiotic Bicyclomycin, thetranscription of several GIs (known as K-islands in E. coli K-12MG1655) significantly increased (Cardinale et al., 2008). These stud-ies also revealed an under-representation of ITs in the sameK-islands. Yet another study, treatment of E. coli K-12 MG1655 withsublethal dosage of Bicyclomycin followed by ChIP-chip analysisshowed several regions on the chromosome where RNAP could lo-calize only in presence of Bicyclomicin (Peters et al., 2009). The in-ference was that Bicyclomycin specifically inhibited Rho in thesecells, thus allowing RNAP to transcribe into regions where Rhowould have caused termination in absence of the antibiotic (Peterset al., 2009). These genomic regions, named Bicyclomycin SensitiveRegions (BSRs), are thus sites where Rho-dependent terminationwould normally occur. The study identified 23 BSRs which weredownstream of K-12-specific genes (belonging to K-islands) or pro-phage DNA. We analyzed the IT profile of these BSRs and foundthat they have an under-representation of ITs and hairpins. Of the23 BSRs that are downstream of the GIs, there was not a single IT oreven a stable hairpin-forming sequence in 16 (70%) of them (Supple-mentary Table S1). Thus, ITs are under-represented in those regionsof E. coli genome where Rho is functioning. In fact, the scarcity of ITsseems to have been compensated by the action of Rho (Cardinale etal., 2008). Hence, Rho is most likely to terminate transcription at

the ends of genes where ITs are absent as these are the only mecha-nisms of termination known in bacteria. This would mean that theintrinsic DIT of the GIs of any genome could be a pointer of Rho activ-ity at such genomic islands. In other words, if the DIT of GI(s) is lowerthan the DIT of the whole genome, then Rho-dependent terminationis probably an important mode of regulation in these GI(s). Hence,we selected representative genomes from different phyla and clas-ses, for which information about GIs was available, and analyzedtheir IT profiles using the algorithm, GeSTer, which detects both ca-nonical and non-canonical ITs (Mitra et al., 2009; Mitra et al., 2010;Unniraman et al., 2002). If the assumption that GIs across bacteriahave extensive Rho-dependent termination is correct, we should ob-serve a consistent trend of decreased presence of ITs in GIs in differ-ent species. Our sample included well characterized prophages,cryptic phages and other kinds of GIs.

3.2. GIs of other E. coli strains are poor in ITs

The importance of Rho-dependent termination in GIs of E. coliwasbased primarily on experiments in E. coli K-12 MG1655. In particular,the paucity of ITs in GIs was shown only for the K-islands of E. coliK-12 (Blattner et al., 1997; Cardinale et al., 2008). At first, we ensuredthat the results reported for the K-islands of E. coli K-12 could also beobtained using GeSTer. Tabulation of the ITs in 42 K-islands(Cardinale et al., 2008) showed that indeed, there was ~50% reduc-tion in DI. DIT in these GIs was only 21.9% as compared to the wholegenomic DIT of E. coli K-12 of 41.7%. Thus, although we had used a dif-ferent algorithm, these results were consistent with the previousstudy. Next, we considered another “model” strain, E. coli 0157:H7EDL933, which also houses several GI, collectively called O-islands(OIs). As with E. coli K-12, the OIs of this genome also show enhancedtranscription after bicyclomycin treatment (Cardinale et al., 2008).Hence, the IT profile of 11 OIs—OI-7, 8, 9, 35, 36, 43, 44, 45, 47, 48and 50 (consisting of a total of 616 genes i.e.11.8% of genome) ofE. coli 0157:H7 EDL933 was analyzed. The major criterion forselecting these OIs was that they all were relatively large GIs. Thelargest among them, OI-43, encoded for 106 genes while the smallest,OI-35, contained 15 genes. Additionally, in order to assess the regionsannotated as resident phages, we selected a prophage (OI-45) andfour representative cryptic phages. Out of 616 genes from these 11O-islands, only 135 genes have an IT immediately downstream.Thus, as observed in E. coli K-12, the number of IT is distinctly lower(DIT=21.9%) in these islands as compared to the genomic DIT of36.6% (Fig. 1A). A closer examination into the IT profiles of the indi-vidual OIs showed that large stretches of genes are devoid of anyITs. Also, as reported in E. coli K-12, we note that many genes occurin series on the same strand and most of these genes, including thegene at the 3′ end of the series, often lack ITs (Cardinale et al.,2008). If these serial gene clusters are operons, then it seems likelythat they lack an IT downstream. In addition, ITs are absent for mostof the genes that are at the 5′ or 3′ ends of the OIs. Lack of identifiableITs hints at the possibility that Rho-dependent termination is proba-bly the major termination mechanism in these OIs.

The genomes of two other strains of E. coli – enteropathogenic E. coli234869 (Iguchi et al., 2009) and uropathogenic E. coli CFT073 (Lloyd etal., 2007) – code for several experimentally characterized pathogenicityislands. The total number of GI genes identified in E. coli 234869 is 493.Besides prophages, these GIs also include the LEE island that has been im-plicated in virulence. Similarly, the CFT073 strain houses the well-knownislands – PAI-II, PAI-III and PAI-CFT073-serX – that encode a total of 299genes (Lloyd et al., 2007, 2009). The DIs of these islands show that thereis a similar decrease in abundance of ITs. The DIT of the islands were19.9% and 20.1% for strains 234869 and CFT073 respectively i.e. between50 and 58% of the genomic values (Fig. 1B, Supplementary Fig. 1A). A de-tailed analysis of the two islands— PAI-II fromstrain CFT073 and LEE fromstrain 234869 for the presence of ITs in relation to the genomic

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Fig. 1. Comparison of IT abundance for GIs of E. coli strains (A) 0157:H7 EDL933 and (B) enteropathogenic 234869. Density of ITs (DIT) refers to the fraction of genes which has anidentified IT downstream of its stop codon. The DIT values for whole genome, the GIs and important pathogenicity islands such as LEE and prophages are provided. (C) Occurrenceand distribution of ITs in the PAI-II island of uropathogenic E. coli strain CFT073. Genes of + and − strands are represented as arrowheads. White, genes which have an IT imme-diately downstream; Gray, rest of the genes. Genes and gene-clusters which do not have an IT downstream are underlined.

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organization reaffirms the observations. PAI-II has 74 genes and can beconsidered divisible into 17 gene clusters (Fig. 1C). Of these, ITs are totallyabsent in case of 11 clusterswhile only three clusters have ITs at the ends.The IT profile of the LEE island is analogous. Of the nine gene clusters inthe LEE island, six clusters have no IT at all (Supplementary Fig S2). Thisincludes the clusters that are at the two ends of the LEE island, suggestingthat Rho-dependent termination may also prevent read-throughtranscription into and out of the island. Thus, both the PAI-II and LEEislands are poor in ITs, with skewed distribution suggesting thatRho-dependent termination is a significant regulator of these GIs. Thus,it seems that GIs across various E. coli strains are poor in ITs, and, as exper-iments have shown in E. coli K-12, are most likely “hotspots” forRho-dependent termination.

3.3. GIs in other γ-proteobacteria have a dearth of ITs

Salmonella enterica serovar Typhimurium (LT2 isolate) has a ge-nome that encodes several prophages, pathogenicity islands andphage remnants. We considered two pathogenicity islands SPI-I(Lostroh and Lee, 2001) and SPI-II (Hensel et al., 1997), four pro-phages and a phage remnant region (4422192–4438335 bp). Similarto the results described above with the E. coli strains, both SPI-I andSPI-II showed very low DIT of 10.4% and 6.8% respectively, comparedto the genomic DIT of 37.2% (Fig. 2A). Analysis of four representative

prophage regions – Gifsy-1, Gifsy-2, Fels-1 and Fels-2 also showedthat the DIT values are consistently lower – between 40 and 60% of thegenomic value. To ascertain that the paucity of ITs was observableonly within the GIs, andwas not a general feature of that part of the ge-nome, we resorted to a “neighboring region” approach. We analyzedthe DIT (Density of Intrinsic Terminators) value of genomic stretchesimmediately adjacent to a GI. The stretch considered was very similarin total number of genes to the GI, but was part of the “core genome”.Thus, it served as a “control experiment” for the in silico analysis. Theabundance of ITs in a 42-genes stretch (STM2644-2693) (DIT=42.8%)was in sharp contrast to the neighboring 46-gene Fels-2 prophagewhich has a DIT of 17.4%. Such “neighborhood analysis” revealed similartrends in other genomes.

Another γ-proteobacterium, Pseudomonas aeruginosa PA-14, harborsthe large PAP-1 island (Battle et al., 2009), shown to be important for vir-ulence. GeSTer analysis showed that the PAP-1 island has only 11 ITs al-though it consists of 114 genes. This means that its DIT is only ~34% ofthe genomic value. A closer inspection of the PAP-1 island (Fig. 3A) indi-cated the absence of ITs at either end of the island. Also, only two of the 16gene-clusters in the PAP-1 island are probably terminated with an IT.Thus, Rho seems to be the major effector of transcription termination inthis GI. The results are in congruence with the initial report in E. coliK-12 and suggest that Rho-dependent termination is indeed a strongregulator at GIs in γ-proteobacteria.

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Fig. 2. ITs profiles inGIs of diverse bacteria. DIT values of genomic islands andprophages of (A) Salmonella enterica serovar Typhimurium(LT2 isolate), (B) Bordetella petrii , (C)Helicobacterpylori and (D)Mycobacteriumabscessus. For each of the four genomes, a “control”, non-GI regionwith comparable number of geneswas selected from the immediate neighborhoodof aGI.In all 4 representative cases, the control region shows a much higher DIT value.

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3.4. IT profiles of α-, β- and ε-proteobacteria species

Next, extending the study beyond γ-proteobacteria, the geno-mic islands of 3 representative proteobacteria – Bordetella petrii(β-proteobacteria), Helicobacter pylori (ε-proteobacteria) and Brucellamelitensis (α-proteobacteria) – were analyzed.

B. petrii, an environmental Bordetella species (Lechner et al., 2009)has 7 large genomic islands (GI-1 to −7) and 2 prophages, encodingfor a total of 1150 genes. In line with the previous results, the GIs of B.petrii also have a lesser number of ITs (~43% of genomic average). If a“control” region of 90 genes just upstream of GI-7 (encodes 87 genes)is considered, it has a DIT of 33.3%, in sharp contrast to GI-7's DIT of11.5% (Fig. 2B). Also, the two prophage regions in the B. petrii havethe lowest DIT.

H. pylori 26695 strain has aDIT of 14.7% (Mitra et al., 2009). However,the DIT of the 27-gene encoding cag island (Blomstergren et al., 2004) is7.4% i.e. 50% of the genomic value (Figs. 2C, 3B). It is noteworthy that al-though absolute values of ITs are lower in this species when comparedto others analyzed, the trend of lower DIT in GIs is consistent across dis-tant species. In contrast, a “control” region of 35 genes immediately up-stream of cag island showed a DIT of 13.9%, very close to the genomicaverage. In case of the pathogenicα-proteobacteria, B. melitensis, a com-parison between the genomic DIT (27.9%) and that of the genomicislands (16%) (Supplementary Fig. 1B) revealed a consistent trend ob-served in other proteobacterial species.

3.5. ITs in the genomic islands in other bacterial phyla

Several actinobacteria genomes sequenced so far also have their shareof GIs. A functional Rho homologue has been reported for Micrococcusluteus, (Nowatzke et al., 1997) Streptomyces lividans and Mycobacteriumtuberculosis (Kalarickal et al., 2009). Thus, it is possible that Rho couldplay a similar “silencing of xenogenic DNA” role in actinobacteria. Forthe present analysis, three genomes were selected — M. tuberculosis,Mycobacterium abscessus and Corynebacterium diphtheriae. GIs havebeen recently identified in the M. tuberculosis H37Rv genome (Becq etal., 2007). The search identified only 36 ITs immediately downstreamof the 454 GIs' genes in M. tuberculosis H37Rv (Supplementary Fig.1C). Also, several “large islands”, notably, Rv739-750, Rv2954-2961,Rv3081-3089, Rv3108-3227 and Rv298-303 did not have a singleflanking or internal IT. The islands Rv0057-0080 (Fig. 3C) andRv0595-0614 had several gene clusters with no IT at their 3′ ends. Theexperimentally characterized genomic island Rv0986-0988 (Rosas-Magallanes et al., 2006) had no IT either. Thus, even for a bacteriumwhich has a distinctly low abundance of ITs (DIT=11.9%), the GIs,which comprise ~10% of the genome, show further decrease in the ITcontent (DIT=7.7%).

In M. abscessus (Ripoll et al., 2009), the causative agent of Buruliulcer, a similar pattern is observed (Fig. 2D). Furthermore, if theGIs of M. abscessus are divided into prophage and non-prophage re-gions, then the three prophages show a further reduction in the

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Fig. 3. Organization and prevalence of genes and ITs in the (A) PAP-I island of Pseudomonas aeruginosa, (B) cag island of Helicobacter pylori and (C) Rv0057-0080 island ofMycobacterium tuberculosis. Genes of + and − strands are represented as arrowheads. White, genes which have an IT immediately downstream; Gray, rest of the genes. Genesand gene-clusters which do not have an IT downstream are underlined.

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number of ITs. In contrast, a “control” region consisting of similar num-ber of genes (MAB0198-0220) immediately upstream of the prophage,MAB0221-0242, has a much larger number of ITs. An analogous situa-tion is seen in the case of the non-mycobacterial actinomycete,C. diphtheriae 13129 (Cerdeno-Tarraga et al., 2003). The two knownprophages of C. diphtheriae are the poorest with respect to the numberof ITs (Supplementary Fig. 1D).

3.6. Fewer operons within GIs have an IT downstream

It can be argued that the differences in DIT between GIs and “core”regions of a given genome are a function of their operonic content. Tocheck this possible scenario, we used the DOOR (Mao et al., 2009),which is considered a reliable database for operon prediction(Brouwer et al., 2008). For any genome, the complete set of transcrip-tion units (TU), that includes both multigenic operons and single-geneTU, can be obtained from DOOR. This data allowed us to ascertain thenumber of TUs in some of the GIs analyzed from diverse species andalso how many of those TUs have an IT downstream. Although the re-sults in this case are obtained from two prediction systems, DOOR andGeSTer are among the most reliable databases available, and so errorsare likely to be minimal. The results show that fewer TUs belongingGIs have an IT downstream, as compared to the genomic estimates(Supplementary Table 2). The lack of ITs at the 3′ ends of many TU inthese GIs indicates that these TU are employing Rho-dependenttermination.

3.7. ITs in the GIs of Bacillus subtilis and Staphylococcus aureus, twospecies with low expression of Rho

B. subtilis, a firmicute, has been shown experimentally to havevery low intracellular levels of Rho, constituting about 0.004% ofthe total cellular soluble protein (Ingham et al., 1999). In contrast,the level of Rho is ~0.15% of the total protein in E. coli, and evenhigher levels of expression of Rho is seen in mycobacteria (Mitra etal., unpublished observations). The non-essential nature of Rho inB. subtilis has been demonstrated by the fact that B. subtilis growswell in the presence of Bicyclomycin, the specific inhibitor of Rho al-though the antibiotic inhibits all known Rho homologues, includingB. subtilis Rho in vitro. Moreover, in other firmicutes such as S. aureusand Streptococcus species, Rho is non-essential (Washburn et al.,2001) or has been lost (Mitra et al., 2009), illustrating the limited im-portance of Rho in firmicutes. Not surprisingly, the firmicutes arespecies' with the highest incidence of ITs (de Hoon et al., 2005;Mitra et al., 2009). Since Rho action seems to be predominant wher-ever there is a lack of ITs, as a corollary, in species with decreasedlevels of Rho, not only “core genome” regions but also genomicislands would employ a larger number of ITs for regulation of geneexpression. Indeed, searching the GIs of B. subtilis 168 genome(Westers et al., 2003) with GeSTer confirms this hypothesis. TheDIT for GIs is 32.7% while the genomic DIT is 41.3%. Similarly, the ge-nomic DIT of S. aureus is 33.3%, while that of three representative GIs(including a prophage and TSST-pathogenicity island) is 25%. Thus,

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of all the species analyzed, the GIs of B. subtilis and S. aureus have thehighest number of ITs. It is most likely that the overall increased de-pendence of B. subtilis and S. aureus on intrinsic termination is mir-rored in its GIs as there is insufficient Rho for efficient inter- andintragenic termination. Such species could be employing othermechanisms such as R-M systems or nucleoid-associated proteinsto silence spurious expression of GIs. For example, the sequences ofGIs often have a GC-content that is lesser than that of the host ge-nome, allowing NAPs to selectively silence such regions (Gordon etal., 2010; Navarre et al., 2006).

3.8. Dearth of ITs in GIs may facilitate Rho-dependent termination

A simple explanation to the observation, consistent with experi-mental data, is that these GIs are “hotspots” for Rho-dependent ter-mination (Cardinale et al., 2008; Peters et al., 2009). A trans factorsuch as Rho is probably advantageous over cis-acting intrinsic termi-nation in the context of GIs. Rho initiates termination by first loadingonto a stretch of nascent RNA called the rut (Rho utilization) site(Richardson, 2003; Richardson and Richardson, 1996). In the fewRho-dependent terminators that have been experimentally charac-terized (Ciampi, 2006) the rut site is C-rich but has no consensus se-quence. Thus, C-rich sequences cannot be termed as specific sites andnot all C-rich sequences are Rho sites. However, Rho also binds toother RNAs as well, and the recent genome-wide studies on Rhohave not identified any degenerate sequence at Rho-dependent ter-mination sites. Infact, the lack of a conserved sequence in the rutsite could well enhance Rho's ability to carry out both intergenic ter-mination as well as intragenic termination of any gene provided it hasaccess to a sufficient length of naked RNA (Ciampi, 2006; Faus andRichardson, 1990; Gowrishankar and Harinarayanan, 2004) (Fig. 4).Additionally, many of these xenogenic genes have a relatively poorcodon adaptation index. Hence, it is likely that the leading ribosomeactually lags far behind the transcribing RNAP allowing Rho to bindto the naked RNA in between the RNAP and the ribosome and causetermination (Richardson, 2006) (Fig. 4). Thus, Rho is uniquely suitedto be primary mediator for prematurely terminating transcription of

Transcription-translation coupled within coding region of highly expressed gene:no intragenic Rho-dependent termination

Poor codon adaptation index of HGT gene; transcription-translation coupling impaired within coding region leading to intragenic Rho- dependent termination

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Fig. 4. Schematic depicting how horizontally transferred, poorly-expressing genes may be sand translation. Generally, in case of genes from the “core genome” transcription–translatioIn contrast, the relatively poor codon adaptation index of a horizontally transferred gene cautermination (C). Subsequently, if the coding sequence of the GI gene gets “adapted”, then i

genes encoded in GIs. Since Rho is functioning, there is no evolution-ary constraint in favor of ITs at these GIs.

However, there are two “limitations” to Rho's mechanism, andboth are based on its limited ability to bypass a hairpin structure onthe transcript. An intervening double stranded RNA stem can preventE. coli Rho's ability to translocate along the RNA towards the RNAP(Steinmetz et al., 1990). Additionally, Rho cannot terminate RNAPwhen the latter has been paused by a class I pause hairpin, (Duttaet al., 2008). The exact mechanism of how a pause hairpin inhibitsRho-dependent termination is unclear. However, it has also beenshown recently that Rho employs an allosteric mechanism to causetermination (Epshtein et al., 2010). Rho interacts with the lid andother domains in the exit channel region of RNAP to transmit an in-hibitory signal to the active center of RNAP. β′ domains extendingfrom the lid and other neighboring parts of the β′ clamp probably me-diate signals from Rho (or Rho-RNA complex) to the catalytic site.

The pause hairpin formed in the exit channel uses the β and β′ do-mains contacted by Rho to transmit a pause-inducing signal to the ac-tive site of RNAP (Toulokhonov and Landick, 2003; Toulokhonov etal., 2001, 2007). Thus, when Rho encounters RNAP paused at a hair-pin, it is most likely that the RNAP domains that Rho would haveused to transduce a terminating signal are either occluded by thehairpin or, alternatively, are in a conformation that is unresponsiveto the factor (Supplementary Fig S3). The paused RNAP would how-ever, resume elongation after a specific time. But, the already formedhairpin that is now extruded from the RNAP exit channel could stillimpede translocation of Rho. Thus, presence of sequences whichhave potential to form hairpins would effectively reduce the efficien-cy of Rho-dependent termination and increase the probability ofRNAP completing the transcription of toxic or unnecessary genes ofGIs. Since Rho-dependent termination seems to be a GI-silencingmechanism it is possible that there could be progressive selectionagainst hairpin-encoding sequences to facilitate Rho action. In otherwords, Rho's silencing action at the various GIs may be facilitated bythe lack of structured RNA moieties like intrinsic terminators andhairpins which makes the RNA unstructured and more suitable as asubstrate for Rho. Moreover, since these regions are not part of the

No transcription-translation coupling after stop codon: intergenic Rho-dependent termination

Improved codon adaptation index of HGT gene; transcription and translation are now coupled within coding region: no intragenic Rho-dependent termination

stop codon

stop codon

B)

D)

Rho interacts with RNAP: termination

ubjected to intragenic, Rho-dependent termination due to uncoupling of transcriptionn coupling allows only intergenic, but not intragenic Rho-dependent termination (A,B).ses uncoupling of transcription and translation favoring even intragenic Rho-dependentntragenic Rho-dependent termination is hindered resulting in gene expression (D).

30 40 50 60 70 800

20

40

60

GC%

% H

airp

ins/

Gen

esn=27

Fig. 5. Inverse correlation between genomic (Hairpins/genes)% and genomic GC con-tent. The sample (n=27) include all the species whose GIs have been analyzed inthis study and also representative species from all major phyla. For each phyla therewere at least one species with low-, intermediate- and high- genomic GC content,and all species had an identifiable Rho gene.

227A. Mitra, V. Nagaraja / Gene 508 (2012) 221–228

core genome it is easier to select against them. However, selectionagainst hairpins by substitution or deletion is also likely to delete asignificant fraction of the ITs since all of them consist of a hairpin. Inthis scenario, their removal is not detrimental as these stretches ofGIs are now regions where there is efficient Rho-dependent termina-tion. In effect, Rho would have functionally replaced ITs in theseregions.

Two pieces of evidence – both of which focus on the mutual exclu-siveness of Rho-dependent terminators and ITs/hairpins – seem tocorroborate the above model. Firstly, as described earlier, theBicyclomycin-sensitive regions (BSRs) of E. coli K-12 MG 1655 ge-nome (Peters et al., 2009) have an under-representation of ITs andpotential hairpins, especially the BSRs that are downstream of K-12specific and prophage DNA (Supplementary Table S1). Secondly,there is an inverse correlation between genomic GC content and theprevalence of ITs in any genome. Additionally, Rho seems to becomemore indispensable in species' as genomic GC content increases.Since experiments have shown that Rho action may be facilitated bythe lack of hairpins (Dutta et al., 2008) it is possible that genomes,which predominantly rely on Rho for termination could have an over-all under-representation of hairpins – both ITs and pause hairpins – inthe regions downstream of the genes. This would happen in both GIsand in core genomic regions, as Rho is a global regulator. To assay this,we determined the total number of hairpins for a sample of bacterialgenomes (n=27) and computed the genomic (hairpins/genes) ratiofor these genome (Fig. 5). The results show that as the genomic GCcontent increases, the genomic (hairpins/genes) value tends to de-crease. The results are in harmony with the fact that most bacteriawhich lack Rho have AT-rich genomes (eg., mycoplasma, many strep-tococci) (Mitra et al., 2009), while Rho seems to be indispensable inspecies with high GC content (eg., Caulobacter crescentus, M. luteus,M. tuberculosis, Steptomyces sp.). In other words, in bacteria whereRho-dependent termination is more important, the absolute numberof stable hairpins in intergenic regions decreases across the entire ge-nome, possibly to favor Rho-dependent termination. Such a situationwould also be consistent with the lack of ITs in GIs across differentgenomes.

3.9. Intergenic, but not intragenic, Rho-dependent termination couldfunction efficiently in expressing GIs

As mentioned earlier, Rho can effect intragenic termination withinthe coding region of a poorly translated gene because transcription–translation coupling is inefficient, allowing the factor to access tonascent RNA and RNAP (Fig. 4). However, if a GI gene that has been

silenced in the past by intragenic Rho-dependent termination is now in-corporated into the cellular machinery, selection is likely to ensure thatthe gene's codon adaptive index is similar to that of the cell. In that case,transcription–translation coupling would now function efficientlypreventing intragenic termination by Rho, and ensure gene expression.However, Rho-dependent termination would still continue to be thepreferred mode of termination, once the stop codon has been crossed(Fig. 5). Thus, Rho is likely to be the default mode of intergenic termina-tion formost GIs, irrespective ofwhether they are silenced or expressedacross species. This could explain why ITs are rare even in genomicislands that are known to express and carry out defined functionssuch as SPI-1, SPI-2, LEE, PAI-II, and cag.

4. Conclusion

Once a genomic island gets integrated into a genome, multiplecheckpoints are likely to ensure that expression from its genes is si-lenced or stringently regulated to prevent any toxicity. Cis factorslike ITs are of limited effectiveness in such situations as they canonly function when sequences that encode them are “strategically”inserted into the GI. In contrast, a trans factor like Rho protein ismore effective in bringing about termination as it has lesser sequenceconstraints and can effectively sense uncoupling of transcription andtranslation. Hence, Rho-dependent termination is likely to be moreeffective in regulating transcription from any xenogenic DNA that en-ters the genome. Consequently, in stretches of the genome wherethere is active Rho-dependent termination (such as GIs), ITs notonly become functionally redundant, but experimental evidencehints that they may also hinder efficient Rho-dependent termination.Hence, over evolutionary timescales, these regions could undergo aselection against such RNA hairpins. Since our analysis is a snapshotin an evolutionary time-scale, a uniform decrease of ITs in GIs acrossdifferent phyla is unlikely to be observed for various reasons. Bothcoding regions and non-coding regulatory elements of GIs are likelyto be subjected to differential selection pressures. Individual GIscould have initially “entered” the host genome with different cohortsof ITs at different time points and varied time spans would haveelapsed since their genomic integration. However, the genome analy-sis across diverse species reinforces the experimental evidence thatRho is indeed an important genome sentinel along with restriction–modification systems, Nucleoid Associated Proteins, transcription re-pressors and other factors (proteins, small RNAs) that act at differentstages to silence expression of foreign DNA. Rho's ability to interactwithout exquisite sequence specificity coupled to its property oftranslocating along RNA interacting with RNAP has resulted in a ver-satile component of cellular “immunity surveillance” mechanism.

Supplementary data to this article can be found online at http://dx.doi.org/10.1016/j.gene.2012.07.064.

Acknowledgments

V. N. is a recipient of the J. C. Bose fellowship of the Department ofScience and Technology, Government of India. The work is supportedby the Centre of Excellence for Mycobacterial Research Grant, Govern-ment of India.

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