# Distribution of Genes Encoding Nucleoid-Associated Protein Homologs in Plasmids

Authors: Toshiharu Takeda; Choong-Soo Yun; Masaki Shintani; Hisakazu Yamane; Hideaki Nojiri
Journal: International Journal of Evolutionary Biology
 (2011)

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## Abstract

Bacterial nucleoid-associated proteins (NAPs) form nucleoprotein complexes and influence the expression of genes. Recent studies have shown that some plasmids carry genes encoding NAP homologs, which play important roles in transcriptional regulation networks between plasmids and host chromosomes. In this study, we determined the distributions of the well-known NAPs Fis, H-NS, HU, IHF, and Lrp and the newly found NAPs MvaT and NdpA among the whole-sequenced 1382 plasmids found in Gram-negative bacteria. Comparisons between NAP distributions and plasmid features (size, G+C content, and putative transferability) were also performed. We found that larger plasmids frequently have NAP gene homologs. Plasmids with H-NS gene homologs had less G+C content. It should be noted that plasmids with the NAP gene homolog also carried the relaxase gene involved in the conjugative transfer of plasmids more frequently than did those without the NAP gene homolog, implying that plasmid-encoded NAP homologs positively contribute to transmissible plasmids.


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## Body

## 1. Introduction

Bacterial chromosomal DNA is folded to form a compacted structure, the nucleoid. The proteins involved in folding the chromosome are known as nucleoid-associated proteins (NAPs) [1, 2]. Because of their DNA-binding ability, NAPs can also play an important role in global gene regulation [1, 2]. Each well-known NAP in Enterobacteriaceae may be categorized as a “factor for inversion stimulation” (Fis), “histone-like nucleoid structuring protein” (H-NS), “histone-like protein from Escherichia coli strain U93” (HU), “integration host factor” (IHF), or “leucine-responsive regulatory protein” (Lrp) [1]. Fis is one of the most abundant NAPs in exponentially growing E. coli cells, and its role as a transcriptional regulator has been investigated [3]. H-NS binds DNA, especially A+T-rich regions including promoter regions or horizontally acquired DNA and acts as a global transcriptional repressor [4]. HU and IHF are similar in amino acid sequence level, and both are global regulators [5, 6], although they have distinct DNA-binding activities: HU binds to DNA nonspecifically whereas IHF binds to a consensus sequence [7]. Lrp has a global influence on transcription regulation and is also involved in microbial virulence [8]. In addition to these well-known NAPs, many other NAPs are found not only in Enterobacteriaceae but also in other organisms. For instance, NdpA, a functionally unknown NAP, has been found in Gram-negative bacteria [9]. The MvaT family protein is the functional homolog of H-NS in Pseudomonas bacteria [10].Horizontal gene transfer (HGT), which is mediated by transduction, transformation, and conjugation, plays an important role in the evolution of prokaryotic genomes [11, 12]. Genes acquired by HGT can provide beneficial functions such as resistance to antibiotics and advantages to their host under selective pressures [13]. However, the mechanisms underlying the integration of newly acquired genes into host regulatory networks are still unclear. Recent investigations have shown that some plasmids carry the genes encoding NAP homologs, which play important roles in transcriptional regulation networks between plasmids and host chromosomes and in maintaining host cell fitness. For example, Doyle et al. [14] reported that plasmid-encoded H-NS-like protein has a “stealth” function that allows for plasmid transfer into host cells without disrupting host regulatory networks, maintaining host cell fitness. Yun and Suzuki et al. [15] reported that plasmid-encoded H-NS-like protein can also play a key role in optimizing gene transcription both on the plasmid and in the host chromosome.In this study, we determined the distributions of NAP homologs among plasmids and discussed their roles in the maintenance of plasmid and host cell fitness.


## 2. Materials and Methods

### 2.1. Plasmid Database Collection and Local BLAST Analyses

The completely sequenced plasmid database was downloaded from the NCBI ftp site (ftp://ftp.ncbi.nih.gov/genomes/Plasmids/). Some duplicated sequence data of the same plasmids were removed from the database. Identification of plasmids that contain the genes encoding NAP homologs was performed using the local TBLASTN program (ver. 2.2.24, ftp://ftp.ncbi.nlm.nih.gov/blast/executables/blast+/LATEST/) under strict conditions (i.e., thresholds of 30% identity and 70% query coverage). The complete amino acid sequences of Fis (DDBJ/EMBL/GenBank accession no. AP_003801), H-NS (AP_001863), Hha (AP_001109), HUα (AP_003818), HUβ (AP_001090), IHFα (AP_002332), IHFβ (AP_001542), Lrp (AP_001519), and NdpA (P33920) from E. coli K-12 W3110 and MvaT (AAP33788) from Pseudomonas aeruginosa PAO1 were used as query sequences.


### 2.2. Bacterial Genome Analyses

The complete genome sequences of bacteria were downloaded from the NCBI ftp site (ftp://ftp.ncbi.nih.gov/genomes/Bacteria/). The number of NAP genes on proteobacterial genomes was investigated using the TBLASTN program (http://www.ncbi.nlm.nih.gov/sutils/genom_table.cgi) under strict conditions (i.e., thresholds of 30% identity and 70% query coverage).


### 2.3. Plasmid Classification

Plasmids in the database were classified into six groups according to their source organisms: Gram-negative, Gram-positive, archaeal, eukaryotic, viral, and unclassified. Putative transferability of each Gram-negative plasmid was determined by whether it carried the relaxase gene of each MOB family that Garcillán-Barcia et al. proposed [16]. Instead of using the local PSI-BLAST program (ver. 2.2.24, ftp://ftp.ncbi.nlm.nih.gov/blast/executables/blast+/LATEST/) as described by Garcillán-Barcia et al. [16], we used the local TBLASTN program.


## 2.1. Plasmid Database Collection and Local BLAST Analyses

The completely sequenced plasmid database was downloaded from the NCBI ftp site (ftp://ftp.ncbi.nih.gov/genomes/Plasmids/). Some duplicated sequence data of the same plasmids were removed from the database. Identification of plasmids that contain the genes encoding NAP homologs was performed using the local TBLASTN program (ver. 2.2.24, ftp://ftp.ncbi.nlm.nih.gov/blast/executables/blast+/LATEST/) under strict conditions (i.e., thresholds of 30% identity and 70% query coverage). The complete amino acid sequences of Fis (DDBJ/EMBL/GenBank accession no. AP_003801), H-NS (AP_001863), Hha (AP_001109), HUα (AP_003818), HUβ (AP_001090), IHFα (AP_002332), IHFβ (AP_001542), Lrp (AP_001519), and NdpA (P33920) from E. coli K-12 W3110 and MvaT (AAP33788) from Pseudomonas aeruginosa PAO1 were used as query sequences.


## 2.2. Bacterial Genome Analyses

The complete genome sequences of bacteria were downloaded from the NCBI ftp site (ftp://ftp.ncbi.nih.gov/genomes/Bacteria/). The number of NAP genes on proteobacterial genomes was investigated using the TBLASTN program (http://www.ncbi.nlm.nih.gov/sutils/genom_table.cgi) under strict conditions (i.e., thresholds of 30% identity and 70% query coverage).


## 2.3. Plasmid Classification

Plasmids in the database were classified into six groups according to their source organisms: Gram-negative, Gram-positive, archaeal, eukaryotic, viral, and unclassified. Putative transferability of each Gram-negative plasmid was determined by whether it carried the relaxase gene of each MOB family that Garcillán-Barcia et al. proposed [16]. Instead of using the local PSI-BLAST program (ver. 2.2.24, ftp://ftp.ncbi.nlm.nih.gov/blast/executables/blast+/LATEST/) as described by Garcillán-Barcia et al. [16], we used the local TBLASTN program.


## 3. Results and Discussion

### 3.1. Database Collection and Plasmid Classification by Origin

We downloaded the whole sequences of 2278 plasmids from the NCBI ftp site (April 2010). Duplicated plasmids were removed manually, and the resultant 2260 plasmid sequences were used in this study. To understand what types of plasmids were included in the database, we classified them into six groups according to their source organisms. The database included 1382 Gram-negative, 725 Gram-positive, 81 archaeal, 43 eukaryotic, 1 viral, and 28 unclassified plasmids.


### 3.2. Identification of the Plasmids Containing NAP Gene Homologs

Using the amino acid sequences of well-known NAPs (Fis, H-NS, HU, IHF, and Lrp) and newly found NAPs (MvaT and NdpA), their distributions were surveyed for plasmids using the TBLASTN program. Some plasmids had ORFs showing sequence similarities to both HU and IHF. We adopted the one with the higher E value. Of 2260 plasmids, 155 (7%) contained the gene encoding NAP homolog. Of those, 116 (75%) contained only one NAP gene homolog and 39 (25%) contained more than one NAP gene homolog. No plasmids carried the Fis gene homolog. Twenty-two plasmids carried the H-NS gene homolog, and all of them had a Gram-negative origin (Table1). Sixty-six plasmids had the HU gene homolog; of these, 51 had a Gram-negative origin and 15 had a Gram-positive origin (Table 2). Twenty-seven plasmids (25 with Gram-negative and 2 with Gram-positive origins) carried the IHF gene homolog (Table 3). Forty-eight plasmids (46 with Gram-negative, 1 with a Gram-positive, and 1 with an archaeal origin) carried the Lrp gene homolog (Table 4). Of these, 23 (48%) contained more than one Lrp gene homolog. On the other hand, MvaT and NdpA homologs were encoded on only 3 plasmids, and all of them were of Gram-negative origin (Table 5). Previously reported plasmids that are known to have NAP gene homologs were included in those 155 plasmids. These included R27 (NC_002305) and pHCM1 (NC_003384) [18, 19] with the H-NS gene homolog; pQBR103 (NC_009444) [20] with the HU and NdpA gene homologs; and pCAR1 (NC_004444) [21, 22] with the MvaT, HU, and NdpA gene homologs. These results indicated the adequacy of our search. Because we used NAPs from Gram-negative bacteria as query sequences, it may be reasonable that 136 (88%) of 155 plasmids with the NAP gene homolog belonged to the group isolated from Gram-negative bacteria. Therefore, in further studies we discussed the Gram-negative plasmid group.Table 1
Plasmids containing the gene encoding H-NS homologa.
Plasmid nameAccession no.Source organismLength (nt)G+C content (%)bIdentity (%)cQuery coverage (%)Subject startSubject endClassificationdMOB familye1NC_013972Erwinia amylovora ATCC 499462824350669931292728−pAsa5NC_009350Aeromonas salmonicidasubsp.salmonicida A449155098544699941534−MOBF47991689016483pAsal5NC_009352Aeromonas salmonicidasubsp.salmonicida185365446991228512692−pEA29NC_013957Erwinia amylovora CFBP14302825950669931292728−pEA29NC_005706Erwinia amylovora2818550649929912590−pEC-IMPNC_012555Enterobacter cloacae318782486499109370108969−MOBHpEC-IMPQNC_012556Enterobacter cloacae324503486499109370108969−MOBHpEJ30NC_004834Erwiniasp. Ejp 5562959350669946514250−pEP36NC_013263Erwinia pyrifoliae Ep1/96359095066992504025441−pEP36NC_004445Erwinia pyrifoliae Ep1/963590450649846754280−pET45NC_010699Erwinia tasmaniensis Et1/99446945152933743537809−MOBFpET49NC_010697Erwinia tasmaniensis Et1/99487514436943082131204−pHCM1NC_003384Salmonella entericasubsp.entericaserovar Typhi str. CT18218160486199131861131460−MOBHpK2044NC_006625Klebsiella pneumoniae NTUH-K20442241525067993571736112−plasmid_153kbNC_009705Yersinia pseudotuberculosis IP 317581531404044100139846140265−pLVPKNC_005249Klebsiella pneumoniae219385506799114397114792−pMAK1NC_009981Salmonella entericasubsp.entericaserovar Choleraesuis2084094761996004659645−MOBHpO111_1NC_013365Escherichia coli O111:H- str. 111282046044761998017579774−MOBHpSG1NC_007713Sodalis glossinidiusstr. “morsitans”8330649439725332922−R27NC_002305Salmonella entericasubsp.entericaserovar Typhi180461466199148225148626−MOBHR478NC_005211Serratia marcescens274762466499111747111346−MOBHUnnamedNC_011148Salmonella entericasubsp.entericaserovar Agona str. SL4833797841439576717288−aThis list is the result of a TBLASTN analysis using the amino acid sequence of H-NS as a query under strict conditions (i.e., thresholds of 30% identity and 70% query coverage). Besides these plasmids, pSf-R27 from Shigella flexneri2a str. 2457T was completely sequenced by Wei et al. [17] and encodes the H-NS-like protein Sfh.bAverage G+C content of the plasmid.cReported TBLASTN identity to H-NS.dPlasmid classification according to its source organism (−, Gram-negative plasmid).ePlasmid classification according to its relaxase gene sequence as described by Garcillán-Barcia et al. [16].Table 2
Plasmids containing the gene encoding HU homologa.
Plasmid nameAccession no.Source organismLength (nt)G+C content (%)bIdentity (%)cQuery coverage (%)Subject startSubject endClassificationdMOB familye1NC_006823Aromatoleum aromaticum EbN1207355585599186175185909−1NC_007949Polaromonassp. JS6663604055752996105260786−MOBH1NC_008010Deinococcus geothermalis DSM 11300574127663897550805550545+1NC_008503Lactococcus lactissubsp.cremoris SK1114041343794973210007+MOBP1NC_008242Chelativoranssp. BNC1343931624194133932133678−MOBQ2NC_012529Deinococcus deserti VCD115314317643893269648269899+3NC_012528Deinococcus deserti VCD11539645961409687008957+MegaplasmidNC_007974Cupriavidus metallidurans CH34258008464519913934151393149−MOBVMegaplasmidNC_005863Desulfovibrio vulgarisstr.Hildenborough20230166319855025765−Megaplasmid pDF308NC_013940Deferribacter desulfuricans SSM13085442441100253817253548−Megaplasmid pHG1NC_005241Ralstonia eutropha H16452156624899343060342791−p49879.1NC_006907Leptospirillum ferrooxidans2887858479932813015−MOBQp49879.2NC_006909Leptospirillum ferrooxidans280125548991585815592−MOBQpAH187_270NC_011655Bacillus cereus AH1872700823459100113139112870+pAH820_272NC_011777Bacillus cereus AH8202721453458100153060152791+pAM04528NC_012693Salmonella enterica1582135257991406714333−MOBHpAOVO01NC_008765Acidovoraxsp. JS427268962461006514064871−MOBFpAPA01-011NC_013210Acetobacter pasteurianus IFO 3283-011917995347100154736154467−46993844238708pAR060302NC_012692Escherichia coli1665305357991575516021−MOBHpAsa4NC_009349Aeromonas salmonicidasubsp.salmonicida A4491667495360992684426578−MOBHpAtS4cNC_011984Agrobacterium vitis S4211620594594141245140991−MOBQpAtS4eNC_011981Agrobacterium vitis S46317755741944047640222−MOBQpBc239NC_011973Bacillus cereus Q12392463352100191895192164+pBF9343NC_006873Bacteroides fragilis NCTC 9343365603235921580315558−MOBPpBPHY01NC_010625Burkholderia phymatum STM8151904893624399826527826252−pBPHY02NC_010627Burkholderia phymatum STM8155951085945999862598359−pBtoxisNC_010076Bacillus thuringiensisserovar israelensis1279233252997738277648+pBWB401NC_010180Bacillus weihenstephanensis KBAB44170543459100338347338078+pCAR1NC_004444Pseudomonas resinovorans1990355642999780998075−MOBHpCAUL01NC_010335Caulobactersp. K312336496744999759897329−MOBQpCER270NC_010924Bacillus cereus2700823459100169548169279+pDBORONC_009137Lactococcus lactissubsp.lactisbv. diacetylactis164043537941638716112+pDVUL01NC_008741Desulfovibrio vulgaris DP4198504663198198317198054−peH4HNC_012690Escherichia coli1481055357991406714333−MOBHpG9842_209NC_011775Bacillus cereus G984220948830601008882888559+pH308197_258NC_011339Bacillus cereus H3081.9725848434591008303383302+pHD5ATNC_012752Candidatus Hamiltonella defensa 5AT (Acyrthosiphon pisum)590324545991498115247−MOBPpIP1202NC_009141Yersinia pestisbv. Orientalis str. IP2751829135357991406714333−MOBHplasmid 2NC_007972Cupriavidus metallidurans CH34171459614699125530125261−pMOL28NC_006525Cupriavidus metallidurans CH341714616146995152951798−pMP118NC_007930Lactobacillus salivarius UCC1182424363254995676356497+MOBVpNPUN02NC_010632Nostoc punctiforme PCC 731022549184144997480474538−MOBVpOANT02NC_009670Ochrobactrum anthropi ATCC 491881014915949943270032446−pP91278NC_008613Photobacterium damselaesubsp.piscicida131520525799125918126184−MOBHpP99-018NC_008612Photobacterium damselaesubsp.piscicida150157515799133314133580−MOBHpPER272NC_010921Bacillus cereus2721453458100153060152791+pPMA4326ANC_005918Pseudomonas syringaepv.maculicola4669755429915201786−pPMA4326BNC_005919Pseudomonas syringaepv.maculicola4011055459914571723−pQBR103NC_009444Pseudomonas fluorescens SBW25425094535199182862183128−pR132503NC_012853Rhizobium leguminosarumbv. trifolii WSM1325516088594794300662300916−MOBQpRA1NC_012885Aeromonas hydrophila1439635158991557315839−MOBHpRALTANC_010529Cupriavidus taiwanensis557200604698153542153276−pREB1NC_009926Acaryochloris marina MBIC110173741614746100339743340012−MOBFpREB2NC_009927Acaryochloris marina MBIC1101735608745481005758357852−MOBFpREB3NC_009928Acaryochloris marina MBIC110172731214546100234682234951−MOBF42100243339243608pRL7NC_008382Rhizobium leguminosarumbv. viciae 38411515645848942048420230−MOBQpRLG203NC_011370Rhizobium leguminosarumbv. trifolii WSM2304308747584994141121140867−pRp12D01NC_012855Ralstonia pickettii 12D389779583799321346321080−MOBHpSG2NC_007184Sodalis glossinidius27240454586100729845−pSG3NC_007186Sodalis glossinidius1920151511001381213543−pSN254NC_009140Salmonella entericasubsp.entericaserovar Newport str. SL2541764735357991406714333−MOBHpTiS4NC_011982Agrobacterium vitis S42588245741942735627102−MOBQ40948340883154pTi-SAKURANC_002147Agrobacterium tumefaciens2064795644949576395509−MOBQpVSAL840NC_011311Aliivibrio salmonicida LFI1238835404060993136131627−MOBF58997735077084pYR1NC_009139Yersinia ruckeri1580385157991507015336−MOBHTiNC_003065Agrobacterium tumefaciens str. C58214233574494139735139481−MOBQaThis list is the result of a TBLASTN analysis using the amino acid sequence of HUα or HUβ as a query under strict conditions (i.e., thresholds of 30% identity and 70% query coverage).bAverage G+C content of the plasmid.cReported TBLASTN identity to HU.dPlasmid classification according to its source organism (−, Gram-negative plasmid; +, Gram-positive plasmid).ePlasmid classification according to its relaxase gene sequence as described by Garcillán-Barcia et al. [16].Table 3
Plasmids containing the gene encoding IHF homologa.
Plasmid nameAccession no.Source organismLength (nt)G+C content (%)bIdentity (%)cQuery coverage (%)Subject startSubject endClassificationdMOB familyeAtNC_003064Agrobacterium tumefaciensstr. C58542868573682112654112412−MOBQMegaplasmidNC_012811Methylobacterium extorquens AM11261460683394720582720860−p2META1NC_012809Methylobacterium extorquens AM1378586544952836928635−MOBQpAACI01NC_013206Alicyclobacillus acidocaldarius subsp.acidocaldarius DSM 446917265443806266862432+pACHL01NC_011879Arthrobacter chlorophenolicus A6426858643292408818408546+pALVIN02NC_013862Allochromatium vinosum DSM 180399295360981090210627−pAph01NC_013193Candidatus Accumulibacter phosphatis clade IIAstr. UW-1167595625695144197144463−MOBPpAph03NC_013191Candidatus Accumulibacter phosphatis clade IIAstr. UW-13769559589754125140−pAtK84bNC_011990Agrobacterium radiobacter K841846685938865410953855−MOBQpAtK84cNC_011987Agrobacterium radiobacter K84388169574393340807340532−46931032710052pAtS4bNC_011991Agrobacterium vitis S41304355647974488045152−MOBQpBBta01NC_009475Bradyrhizobiumsp. BTAi122882661398666426388−pBFY46NC_006297Bacteroides fragilis YCH46337163435892509825343−MOBPpBIND01NC_010580Beijerinckia indicasubsp.indica ATCC 9039181736563677179816179601−MOBFpCHQ1NC_014007Sphingobium japonicum UT26S1909746336906311163377−pGLOV01NC_010815Geobacter lovleyi SZ771135338924119641468−pM44601NC_010373Methylobacteriumsp. 4-465795165359778067534−pMPOP01NC_010727Methylobacterium populi BJ001251646549931063510375−pMRAD03NC_010514Methylobacterium radiotolerans JCM 2831429856338942677826515−MOBFpMRAD04NC_010517Methylobacterium radiotolerans JCM 2831377436438941076310500−pPRO1NC_008607Pelobacter propionicus DSM 2379202397484194129679129957−pRSPA01NC_009429Rhodobacter sphaeroides ATCC 17025877879684997783519783791−pSWIT01NC_009507Sphingomonas wittichii RW1310228644095106554106820−MOBF36923534135069pTcM1NC_010600Acidithiobacillus caldus651585756892518625449−MOBP, MOBQpXCV183NC_007507Xanthomonas campestrispv. vesicatoriastr. 85-10182572603395138753138490−TiNC_002377Agrobacterium tumefaciens194140554397180164180436−MOBQTi plasmid pTiBo542NC_010929Agrobacterium tumefaciens244978553686209743209489−MOBQ4598187204187479aThis list is the result of a TBLASTN analysis using the amino acid sequence of IHFα or IHFβ as a query under strict conditions (i.e., thresholds of 30% identity and 70% query coverage).bAverage G+C content of the plasmid.cReported TBLASTN identity to IHF.dPlasmid classification according to its source organism (−, Gram-negative plasmid; +, Gram-positive plasmid).ePlasmid classification according to its relaxase gene sequence as described by Garcillán-Barcia et al. [16].Table 4
Plasmids containing the gene encoding Lrp homologa.
Plasmid nameAccession no.Source organismLength (nt)G+C content (%)bIdentity (%)cQuery coverage (%)Subject startSubject endClassificationdMOB familye1NC_008688Paracoccus denitrificans PD1222653815674192252075251623−429346421846467336966393416398113785110140109724ANC_009007Rhodobacter sphaeroides 2.4.11140456939933024129789−MOBFBNC_007488Rhodobacter sphaeroides 2.4.11141787043968186181385−bglu_1pNC_012723Burkholderia glumae BGR1133591613690124017123577−MegaplasmidNC_008043Ruegeriasp. TM1040821788594184143820144233−41916872576877063691734136733690MegaplasmidNC_007974Cupriavidus metallidurans CH34258008464448811712451170814−MOBV409111697021169256389715867261586250MegaplasmidNC_006569Ruegeria pomeroyi DSS-3491611633688356303355869−MOBCMegaplasmidNC_007336Ralstonia eutropha JMP134634917613593377503377045−p42eNC_007765Rhizobium etli CFN 42505334623471255037255384−p42fNC_007766Rhizobium etli CFN 42642517614588436907437341−4391406350405901418549138349179939952106342110983996199426199899pAB510aNC_013855Azospirillumsp. B5101455109685788274908275342−4495979549980013329411803351179874pAB510bNC_013856Azospirillumsp. B510723779674484471830472243−3294318139318600pAB510cNC_013857Azospirillumsp. B510681723674585408064407645−34913638536834pAB510dNC_013858Azospirillumsp. B510628837684479472768472379−409032318432274137872814382818663085619027618623pAtS4eNC_011981Agrobacterium vitis S4631775573087460443460871−MOBQ3474425247424888pBPHY01NC_010625Burkholderia phymatum STM815190489362468511536081154027−pBPHY02NC_010627Burkholderia phymatum STM815595108594191271795271346−pCNC_010997Rhizobium etli CIAT 6521091523614688617696618130−MOBQ4290609059608619399541773841820242797148047151933993406570407025pCAUL01NC_010335Caulobactersp. K31233649673489182479182042−MOBQpEST4011NC_005793Achromobacter denitrificans769586258884122440793−MOBP58883423333802pGMI1000MPNC_003296Ralstonia solanacearum GMI1000209450967439817379581738437−4693822030821572pHV4NC_013966Haloferax volcanii DS2635786623371401763401410ArchaeapIJB1NC_013666Burkholderia cepacia994486358887490775338−MOBPpK2044NC_006625Klebsiella pneumoniae NTUH-K2044224152503390194643195086−pLVPKNC_005249Klebsiella pneumoniae2193855033904623646679−pMLaNC_002679Mesorhizobium loti MAFF303099351911593293185603185148−3089207314206877pMLbNC_002682Mesorhizobium loti MAFF3030992083156037932463224177−pNGR234aNC_000914Rhizobiumsp. NGR234536165584170197189196845−MOBQ3089188867188430pNGR234bNC_012586Rhizobiumsp. NGR2342430033624690656547656107−MOBQ458566749466791343901038020103846344856827966832153896240084924013194479709104708715418928336287613389110890011093373690703213702764327711129531112582pPNAP04NC_008760Polaromonas naphthalenivorans CJ2143747593590142511142068−pR132501NC_012848Rhizobium leguminosarumbv.trifolii WSM1325828924604788234905234471−MOBQ448638633838676039936455426450874279147165146776pRALTANC_010529Cupriavidus taiwanensis557200603891465839465393−pRHL1NC_008269Rhodococcus jostii RHA11123075653691854207854656+3384783666783253pRL12NC_008378Rhizobium leguminosarumbv. viciae 3841870021614688599116598682−MOBQ4388658287658718399345601451464279450080449691pRL8NC_008383Rhizobium leguminosarumbv. viciae 38411474635933877076370344−MOBQpRLG201NC_011368Rhizobium leguminosarumbv. trifolii WSM23041266105604589917573917136−MOBQ4485419984241744794730394726504093116214611616914093115093911504843288707587707162pRSKD131ANC_011962Rhodobacter sphaeroides KD131157345704296148295147819−pRSKD131BNC_011960Rhodobacter sphaeroides KD1311033557039939840097948−pRSPA01NC_009429Rhodobacter sphaeroides ATCC 170258778796840903130930866−3988659383658952pRSPH01NC_009040Rhodobacter sphaeroides ATCC 17029122606703993118088118540−pSMED01NC_009620Sinorhizobium medicae WSM4191570951614077143180143557−MOBQ3489574284573847pSMED02NC_009621Sinorhizobium medicae WSM4191245408604291556486556932−MOBQ409184232484275831872234521917pSMED03NC_009622Sinorhizobium medicae WSM419219313604695105044105508−pSmeSM11aNC_013545Sinorhizobium meliloti1441706046967044970922−MOBQpSymANC_003037Sinorhizobium meliloti 1021135422660438910606991060262−MOBQpSymBNC_003078Sinorhizobium meliloti 10211683333623890440778440335−MOBQ36892955529992pTiS4NC_011982Agrobacterium vitis S42588245742799692097309−MOBQUnnamedNC_011961Thermomicrobium roseum DSM 5159917738663085736739737146−MOBPaThis list is the result of a TBLASTN analysis using the amino acid sequence of Lrp as a query under strict conditions (i.e., thresholds of 30% identity and 70% query coverage).bAverage G+C content of the plasmid.cReported TBLASTN identity to Lrp.dPlasmid classification according to its source organism (−, Gram-negative plasmid; +, Gram-positive plasmid).ePlasmid classification according to its relaxase gene sequence as described by Garcillán-Barcia et al. [16].Table 5
Plasmids containing the gene encoding MvaT or NdpA homologa.
Plasmid nameAccession no.Source organismLength (nt)G+C content (%)bIdentity (%)cQuery coverage (%)Subject startSubject endClassificationdMOB familyeMvaTpCAR1NC_004444Pseudomonas resinovorans1990355661987764077993−MOBHpQBR103NC_009444Pseudomonas fluorescens SBW254250945361969807697717−pWW53NC_008275Pseudomonas putida10792957619884158768−NdpAp0908NC_010113Vibriosp. 0908814134951997973178736−pCAR1NC_004444Pseudomonas resinovorans1990355636989539094395−MOBHpQBR103NC_009444Pseudomonas fluorescens SBW25425094533199161413160400−aThis list is the result of a TBLASTN analysis using the amino acid sequence of MvaT or NdpA as a query under strict conditions (i.e., thresholds of 30% identity and 70% query coverage).bAverage G+C content of the plasmid.cReported TBLASTN identity to MvaT or NdpA.dPlasmid classification according to its source organism (−, Gram-negative plasmid).ePlasmid classification according to its relaxase gene sequence as described by Garcillán-Barcia et al. [16].


### 3.3. Relationships between Plasmid Size and NAP Gene Homolog Distributions

We first compared the sizes of 136 plasmids with NAP gene homologs with those of all 1382 Gram-negative group plasmids. All 1382 plasmids could be divided into 4 groups according to size, small (<10 kb), intermediate (10 to 100 kb), large (100 kb to 1 Mb), and mega (>1 Mb) plasmids. The distribution of the 136 plasmids, each of which had one or more genes encoding NAP homologs, is shown in Figure1(a): none of 415 small plasmids, 34 (5%) of 686 intermediate plasmids, 90 (33%) of 269 large plasmids, and 12 (100%) of 12 mega plasmids carried at least one NAP gene homolog. The average size of the 136 plasmids was larger (364 kb) than that of all 1382 plasmids (83 kb). These results suggest that larger plasmids, especially >100 kb, frequently have NAP gene homologs. Carrying large plasmids may reduce host fitness more than carrying small plasmids because the former have more genes that can disrupt transcriptional networks in the host cell. In addition, large plasmids may have more binding sites for NAPs than small plasmids. Because chromosome-encoded NAPs bind to both chromosomes and plasmids, carrying large plasmids may also result in a reduction in the binding of NAPs to the host chromosome, causing undesirable effects on the host cell. Plasmid-encoded NAP homologs may interact with chromosome-encoded NAPs, coordinately sustain the structure of both chromosome and plasmid, and regulate the transcriptional regulation network [23]. In fact, recent studies have shown that some plasmid-encoded NAP homologs can complement the depletion of chromosomal NAPs and optimize gene transcription both on plasmids and in the host chromosome [14, 15, 24]. Thus, larger plasmids may have NAP gene homologs to maintain host cell fitness. In addition, the average size of the 38 plasmids containing more than one NAP gene homolog was larger (790 kb) than that of the 98 plasmids containing only one NAP gene homolog (199 kb). This suggests that particularly large plasmids have many NAP gene homologs to maintain themselves in the host cell.Size comparison of the Gram-negative plasmids with and without NAP gene homologs. (a) A total of 136 Gram-negative plasmids with one or more NAP gene homologs and 1246 Gram-negative plasmids without NAP gene homologs are shown by black and white bars, respectively. (b) Gram-negative plasmids with each NAP gene homolog are as follows: H-NS, red; HU, blue; IHF, green; Lrp, purple; MvaT, yellow; and NdpA, orange.
(a)(b)Distributions of the NAP genes on proteobacterial genomes were also surveyed using the TBLASTN program. The average size of the completely sequenced bacterial genomes was 3.25 Mb and 1054 NAP genes (100, Fis; 125, H-NS; 236, HU; 247, IHF; 127, Lrp; 119, MvaT; and 100, NdpA) were found in 588 proteobacterial genomes. Frequency of NAP genes in plasmids was higher (1 per 236 kb) than that in proteobacterial genomes (1 per 1.8 Mb), also suggesting that larger plasmids frequently have NAP gene homologs to minimize their negative effects on the host cell.Of the plasmids with the NAP gene homolog, the average size of those with the H-NS gene homolog was relatively small (132 kb) while that of those with the Lrp gene homolog was relatively large (725 kb). The average sizes of those with the other NAP gene homologs were as follows: HU (301 kb), IHF (230 kb), MvaT (244 kb), and NdpA (235 kb) (Figure1(b)). H-NS exists in an oligomeric form and binds to DNA, especially A+T-rich regions, by bridging it [25]. This function may be important for regulating gene expression on relatively small plasmids among those with the NAP gene homolog. The activity of H-NS can also be modulated by Hha-like proteins [26]. Intriguingly, TBLASTN analysis showed that 12 (55%) of 22 plasmids with the H-NS gene homolog also carried gene encoding Hha-like protein although only 65 (5%) of all 1382 plasmids carried Hha-like protein gene (Table 6). This suggests the close relationship of H-NS and Hha-like protein. On the other hand, Lrp exists in dimeric, octameric, and hexadecameric forms and compacts DNA by wrapping it [27]. This distinctive DNA-binding ability may be essential for maintaining the structure of particularly larger plasmids.Table 6
Gram-negative plasmids containing the gene encoding Hha-like proteina.
Plasmid nameAccession no.Source organismLength (nt)G+C content (%)bNAP gene homologIdentity (%)cQuery coverage (%)Subject startSubject endMOB familyd55989pNC_011752Escherichia coli 5598972482465392100259828NR1NC_009133Escherichia coli942895253928719387390MOBFp1658/97NC_004998Escherichia coli1254915155923641936616MOBFp1ESCUMNC_011749Escherichia coli UMN0261223015053925350853311MOBFp2ESCUMNC_011739Escherichia coli UMN0263380942629076827488MOBQp53638_226NC_010719Escherichia coli 536382256834855926761567418MOBFpAPEC-O1-RNC_009838Escherichia coli APEC O12413874650926138961586MOBHpAPEC-O2-ColVNC_007675Escherichia coli18450149559238823685MOBFpAPEC-O2-RNC_006671Escherichia coli10137553539248564659MOBFpBS512_211NC_010660Shigella boydii CDC 3083-94210919465589190719190910MOBFpBS512_33NC_010657Shigella boydii CDC 3083-943310341629028943088pC15-1aNC_005327Escherichia coli923535353928749087687MOBFpCP301NC_004851Shigella flexneri2a str. 301221618465592207828208025MOBFpCROD1NC_013717Citrobacter rodentium ICC168544494756925322053417pCROD2NC_013718Citrobacter rodentium ICC168392654262901552615332pCT02021853_74NC_011204Salmonella entericasubsp.entericaserovar Dublin str. CT_02021853745514962904848248288MOBQpCTX-M3NC_004464Citrobacter freundii894685138712613626294MOBP8946831964064840439pCTXM360NC_011641Klebsiella pneumoniae680185138716455164709MOBP6801831961092710718pCVM29188_146NC_011076Salmonella entericasubsp.entericaserovar Kentucky str. CVM291881468114953921875518558MOBFpEC14_114NC_013175Escherichia coli114222515392113985114182MOBFpEC-IMPNC_012555Enterobacter cloacae31878248H-NS50926049160688MOBHpEC-IMPQNC_012556Enterobacter cloacae32450348H-NS50926049160688MOBHpEG356NC_013727Shigella sonnei702755253926944469641MOBFpEK499NC_013122Escherichia coli1175365353924198542182pEK516NC_013121Escherichia coli644715353923141031213pEL60NC_005246Erwinia amylovora601455138712318723345MOBP6014531963786337654pEntH10407NC_013507Escherichia coli ETEC H10407670945155784342143254MOBFpHCM1NC_003384Salmonella entericasubsp.entericaserovar Typhi str. CT1821816048H-NS47100105911106117MOBHpK2044NC_006625Klebsiella pneumoniae NTUH-K204422415250H-NS, Lrp4585143331143528pK29NC_010870Klebsiella pneumoniae2696744650925932259519MOBHpKF3-70NC_013542Klebsiella pneumoniae700575253921596715770MOBFpKF3-94NC_013950Klebsiella pneumoniae9421952589695969390MOBFpKP187NC_011282Klebsiella pneumoniae 342187922476496110083109877187922428915501344pKPN3NC_009649Klebsiella pneumoniaesubsp.pneumoniae MGH 785781758795259975693056721MOBFplasmid_153 kbNC_009705Yersinia pseudotuberculosis IP 3175815314040H-NS6993633426354215314056924973449931pLVPKNC_005249Klebsiella pneumoniae21938550H-NS, Lrp61971480561478472193854585214828215025pMAK1NC_009981Salmonella entericasubsp.entericaserovar Choleraesuis20840947H-NS471004920849414MOBHpMAS2027NC_013503Escherichia coli426444362901968519491MOBQpO103NC_013354Escherichia coli O103:H2 str. 12009755464955925172751924MOBFpO111_1NC_013365Escherichia coli O111:H- str. 1112820460447H-NS471006692567131MOBHpO111_3NC_013366Escherichia coli O111:H- str. 11128776905055921197512172MOBFpO157NC_013010Escherichia coli O157:H7 str. TW14359946014855927079270989pO157NC_011350Escherichia coli O157:H7 str. EC4115946444855925473554932pO157NC_007414Escherichia coli O157:H7 EDL9339207748559216671864pO157NC_002128Escherichia coli O157:H7 str. Sakai927214855927118371380pO26INC_011812Escherichia coli729465153926660866805MOBFpO86A1NC_008460Escherichia coli120730495592101598101795MOBFpOLA52NC_010378Escherichia coli516024662901211411920MOBQpOU1114NC_010421Salmonella entericasubsp. entericaserovar Dublin3459541629054465252MOBQpOU1115NC_010422Salmonella entericasubsp.entericaserovar Dublin745894962903724637052MOBQpSB4_227NC_007608Shigella boydii Sb227126697475592110688110885MOBFpSE11-1NC_011419Escherichia coli SE111000215056925840758210MOBPpSE34NC_010860Salmonella entericasubsp.entericaserovar Enteritidis329504162902187522069MOBQpSFO157NC_009602Escherichia coli12123950527517091870MOBFpSG1NC_007713Sodalis glossinidiusstr. “morsitans”8330649H-NS489222942491pSG1NC_007182Sodalis glossinidius815534948925621756414pSMS35_130NC_010488Escherichia coli SMS-3-513044051559233643167MOBFpSS_046NC_007385Shigella sonnei Ss046214396455592178363178560MOBFpUTI89NC_007941Escherichia coli UTI89114230515392113993114190MOBFpWR501NC_002698Shigella flexneri221851465592207534207731MOBFR100NC_002134Shigella flexneri2b str. 222942815253928718587382MOBFR27NC_002305Salmonella entericasubsp.entericaserovar Typhi18046146H-NS47100159402159196MOBHR478NC_005211Serratia marcescens27476246H-NS50925942659623MOBHR721NC_002525Escherichia coli755824366903528535091UnnamedNC_011148Salmonella entericasubsp.entericaserovar Agona str. SL4833797841H-NS429313631163aThis list is the result of a TBLASTN analysis using the amino acid sequence of Hha as a query under strict conditions (i.e., thresholds of 30% identity and 70% query coverage).bAverage G+C content of the plasmid.cReported TBLASTN identity to Hha.dPlasmid classification according to its relaxase gene sequence as described by Garcillán-Barcia et al. [16].


### 3.4. Relationships between Plasmid G+C Content and NAP Gene Homolog Distributions

Next, we surveyed the G+C content of the Gram-negative group plasmids with and without NAP gene homologs. The average G+C content of the 136 plasmids with NAP gene homologs was higher (56.4%) than that of all 1382 plasmids (44.8%) (Figure2(a)). Note that the average G+C content of large and mega plasmids (55.0% and 62.9%, resp.) was higher than that of small and intermediate plasmids (44.8% and 40.4%). Considering that larger plasmids frequently had NAP gene homologs, this seems reasonable. Nevertheless, plasmids with H-NS gene homologs had a lower G+C content (48.3%) than did those with other NAP gene homologs, including HU (54.2%), IHF (58.7%), Lrp (62.3%), MvaT (55.6%), and NdpA (52.9%) (Figure 2(b)). H-NS family protein binds A+T-rich regions not only on chromosomes but also on plasmids [15]. Acquisition of a large A+T-rich plasmid with many H-NS binding sites may result in a reduction in the binding of H-NS to the host chromosome and host cell fitness [14]. It is therefore possible that large A+T-rich plasmids may have to supply another H-NS encoded on themselves to minimize the effect on the host cell. On the other hand, although MvaT-family proteins are the functional homolog of H-NS [10, 15], plasmids containing the MvaT gene homolog were not particularly low in G+C content. Although only three plasmids contained the MvaT gene homolog and thus we cannot discuss this interesting phenomenon in detail, the difference between H-NS and MvaT may be derived from their different origin or host bacteria.G+C content comparison of the Gram-negative plasmids with and without NAP gene homologs. (a) A total of 136 Gram-negative plasmids with one or more NAP gene homologs and 1246 Gram-negative plasmids without NAP gene homologs are shown by black and white bars, respectively. (b) Gram-negative plasmids with each NAP gene homolog are as follows: H-NS, red; HU, blue; IHF, green; Lrp, purple; MvaT, yellow; and NdpA, orange.
(a)(b)


### 3.5. Relationships between Plasmid Transferability and NAP Gene Homolog Distributions

Conjugative transfer is an essential function of plasmids, through which they play an important role in bacterial evolution and host cell behavior [11, 12]. Relaxase is an essential protein for plasmid transmission involved in the cleavage of the transferring DNA at the origin of transfer (oriT) site, and plasmids with relaxase genes are thought to be transmissible. Garcillán-Barcia et al. [16] proposed that transmissible plasmids can be classified into 6 MOB families (MOBC, MOBF, MOBH, MOBP, MOBQ, and MOBV) according to the amino acid sequences of 6 prototype relaxase proteins. MOBF and MOBH families are predominantly composed of conjugative plasmids, also called self-transmissible plasmids, and the other 4 families are composed of both mobilizable and conjugative plasmids. Recent studies have reported that plasmid-encoded H-NS family proteins have a “stealth” function and aide horizontal transfer of plasmids [14, 15]. Other NAPs also act as global transcriptional regulators and may regulate expression of genes involved in plasmid transmission. To discuss the relationship between NAP gene homolog distribution and plasmid transferability, we determined the distribution of genes encoding relaxase proteins in Gram-negative plasmids according to the classification by Garcillán-Barcia et al. [16]. Four hundred and nine (30%) of 1382 Gram-negative plasmids carried relaxase genes, and 71 (17%) of those 409 plasmids carried NAP gene homologs. Note that 71 (52%) of 136 plasmids with NAP gene homologs carried relaxase genes. This indicates that plasmids with NAP gene homologs frequently carried the relaxase genes than did those without NAP gene homologs. This phenomenon may be related to the average size of the plasmids. That of the 409 plasmids with relaxase genes was relatively larger (145 kb) than that of all 1382 plasmids (83 kb), corresponding to the fact that larger plasmids frequently had NAP gene homologs.Four hundred and nine plasmids were classified into each MOB family (13, MOBC; 128, MOBF; 29, MOBH; 86, MOBP; 131, MOBQ; and 26, MOBV). Plasmid 1 (NC_008545) was classified into both the MOBC and MOBF families. In addition, the MOBP, MOBQ, and MOBV families were partially overlapped as described by Garcillán-Barcia et al. [16]. Seventy-one plasmids with NAP gene homologs were contained in each MOB family (1, MOBC; 11, MOBF; 20, MOBH; 8, MOBP; 30, MOBQ; and 2, MOBV). Intriguingly, 20 (69%) of 29 MOBH-family plasmids encoded some NAP homologs, and most of them were H-NS or HU (Table 7). The MOBH family was composed of predominantly large conjugative plasmids, such as the IncHI1 group of plasmids, suggesting that HU may also contribute to plasmid transmission as does H-NS. Furthermore, 30 (23%) of 131 MOBQ-family plasmids also contained some NAP gene homologs, and 15 (50%) of those carried Lrp gene homologs (Table 8). The MOBQ family was composed of both mobilizable and conjugative plasmids, such as those of Rhizobium and Agrobacterium, implying that Lrp may also affect plasmid conjugation. In the other MOB families, plasmids containing NAP gene homologs were less than 10% (8%, MOBC; 9%, MOBF; 9%, MOBP; and 8%, MOBV). This phenomenon may also be related to the average size of the plasmids contained in each MOB family. MOBH (220 kb) and MOBQ (198 kb) were larger than MOBC (78 kb), MOBF (117 kb), MOBP (87 kb), and MOBV (149 kb). On the other hand, the average G+C content of all plasmids belonging to each MOB family was as follows: MOBC (52%), MOBF (52%), MOBH (51%), MOBP (53%), MOBQ (54%), and MOBV (46%). No relationship between the distribution of NAP gene homologs of each MOB family and the G+C content of plasmids was found.Table 7
MOBH-family plasmids of Gram-negative origina.
Plasmid nameAccession no.Source organismLength (nt)G+C content (%)bNAP gene homologIdentity (%)cQuery coverage (%)Subject startSubject end1NC_007949Polaromonassp. JS66636040557HU529961052607861NC_008573Shewanellasp. ANA-3278942462NC_007950Polaromonassp. JS66633800760ICEhin1056NC_011409Haemophilus influenzae5939339pAM04528NC_012693Salmonella enterica15821352HU57991406714333pAPEC-O1-RNC_009838Escherichia coli APEC O124138746pAR060302NC_012692Escherichia coli16653053HU57991575516021pAsa4NC_009349Aeromonas salmonicidasubsp.salmonicida A44916674953HU60992684426578pCAR1NC_004444Pseudomonas resinovorans19903556MvaT61987764077993NdpA36989539094395HU42999780998075pEC-IMPNC_012555Enterobacter cloacae31878248H-NS6499109370108969pEC-IMPQNC_012556Enterobacter cloacae32450348H-NS6499109370108969peH4HNC_012690Escherichia coli14810553HU57991406714333pHCM1NC_003384Salmonella entericasubsp.entericaserovar Typhi str. CT1821816048H-NS6199131861131460pIP1202NC_009141Yersinia pestisbv. Orientalis str. IP27518291353HU57991406714333pK29NC_010870Klebsiella pneumoniae26967446plasmid1NC_007901Rhodoferax ferrireducens T11825744754pMAK1NC_009981Salmonella entericasubsp.entericaserovar Choleraesuis20840947H-NS61996004659645pMAQU02NC_008739Marinobacter aquaeolei VT821329053pO111_1NC_013365Escherichia coli O111:H- str. 1112820460447H-NS61998017579774pP91278NC_008613Photobacterium damselaesubsp. Piscicida13152052HU5799125918126184pP99-018NC_008612Photobacterium damselaesubsp.piscicida15015751HU5799133314133580pRA1NC_012885Aeromonas hydrophila14396351HU58991557315839pRp12D01NC_012855Ralstonia pickettii 12D38977958HU3799321346321080pSN254NC_009140Salmonella entericasubsp.entericaserovar Newport str. SL25417647353HU57991406714333pTK9001NC_013930Thioalkalivibriosp. K90mix24025662pYR1NC_009139Yersinia ruckeri15803851HU57991507015336R27NC_002305Salmonella entericasubsp.entericaserovar Typhi18046146H-NS6199148225148626R478NC_005211Serratia marcescens27476246H-NS6499111747111346Rts1NC_003905Proteus vulgaris21718246aThis list is the result of a TBLASTN analysis using the 300 N-terminal amino acid sequence of protein TraI_R27 as a query under strict conditions (i.e., thresholds of 30% identity and 70% query coverage).bAverage G+C content of the plasmid.cReported TBLASTN identity to each NAP.Table 8
MOBQ-family plasmids of Gram-negative origina.
Plasmid nameAccession no.Source organismLength (nt)G+C content (%)bNAP gene homologIdentity (%)cQuery coverage (%)Subject startSubject end1NC_008242Chelativoranssp. BNC134393162HU41941339321336783NC_007617Nitrosospira multiformis ATCC 2519614159503NC_007961Nitrobacter hamburgensis X1412140862AtNC_003064Agrobacterium tumefaciensstr. C5854286857IHF3682112654112412CNC_010542Cyanothecesp. ATCC 511421468538ColE9-JNC_011977Escherichia coli757750DN1NC_002636Dichelobacter nodosus511262F plasmidNC_008036Sphingopyxis alaskensis RB22562854360p11745NC_013546Actinobacillus pleuropneumoniae548638p12494NC_010889Actinobacillus pleuropneumoniae1439333p1ABAYENC_010401Acinetobacter baumannii AYE564435p1META1NC_012807Methylobacterium extorquens AM14419568p1METDINC_012987Methylobacterium extorquens DM414150465p2007057NC_011897Salmonella entericasubsp.entericaserovar Bovismorbificans427047p2ABSDFNC_010396Acinetobacter baumannii SDF2501435p2ESCUMNC_011739Escherichia coli UMN0263380942p2META1NC_012809Methylobacterium extorquens AM13785865IHF44952836928635p3ABSDFNC_010398Acinetobacter baumannii SDF2492234p42aNC_007762Rhizobium etli CFN 4219422958p49879.1NC_006907Leptospirillum ferrooxidans2887858HU479932813015p49879.2NC_006909Leptospirillum ferrooxidans2801255HU48991585815592pAb5S9NC_009476Aeromonas bestiarum2471654pACRY07NC_009473Acidiphilium cryptum JF-5562958pAgK84NC_011994Agrobacterium radiobacter K844442054pAM5NC_008691Acidiphilium multivorum516158pAMI2NC_010847Paracoccus aminophilus1856362pAMI3NC_013513Paracoccus aminophilus557561pAPA01-030NC_013212Acetobacter pasteurianus IFO 3283-014996154pAPA01-040NC_013213Acetobacter pasteurianus IFO 3283-01320454pAtK84bNC_011990Agrobacterium radiobacter K8418466859IHF38865410953855pAtS4bNC_011991Agrobacterium vitis S413043556IHF47974488045152pAtS4cNC_011984Agrobacterium vitis S421162059HU4594141245140991pAtS4eNC_011981Agrobacterium vitis S463177557HU41944047640222Lrp3087460443460871Lrp3474425247424888pAV2NC_010310Acinetobacter venetianus1513536pBNC_010996Rhizobium etli CIAT 65242911158pBGR3NC_012847Bartonella grahamii as4aup2819236pBS512_5NC_010659Shigella boydii CDC 3083-94511446pCNC_010997Rhizobium etli CIAT 652109152361Lrp4688617696618130Lrp4290609059608619Lrp3995417738418202Lrp4279714804715193Lrp3993406570407025pCAUL01NC_010335Caulobactersp. K3123364967HU44999759897329Lrp3489182479182042pCAUL02NC_010333Caulobactersp. K3117787864pCCK1900NC_011378Pasteurella multocida1022661pCCK381NC_006994Pasteurella multocida1087461pCFPG4NC_011563Candidatus Azobacteroides pseudotrichonymphagenomovar. CFP2414944pCHE-ANC_012006Enterobacter cloacae756060pColE8NC_012882Escherichia coli675151pCROD3NC_013719Citrobacter rodentium ICC168391051pCT02021853_74NC_011204Salmonella entericasubsp.entericaserovar Dublin str. CT_020218537455149pCVM19633_4NC_011093Salmonella entericasubsp.entericaserovar Schwarzengrund str. CVM19633458548pDSHI01NC_009955Dinoroseobacter shibae DFL 1219050660pET09NC_010695Erwinia tasmaniensis Et1/99929947pGDIA01NC_011367Gluconacetobacter diazotrophicus PAl 52745559pGOX3NC_006674Gluconobacter oxydans 621H1454756pHCG3NC_005873Oligotropha carboxidovorans OM513305861pHRM2aNC_012109Desulfobacterium autotrophicum HRM26870942pIGJC156NC_009781Escherichia coli514647pIGMS5NC_010883Escherichia coli675051pIGWZ12NC_010885Escherichia coli407250pISP3NC_013970Sphingomonassp. MM-14339863pJD4NC_002098Neisseria gonorrhoeae742638plasmid1NC_007801Jannaschiasp. CCS18607258pLD-TEX-KLNC_009966Fluoribacter dumoffii6651239pMACNC_006877Acinetobacter baumannii954035pMAS2027NC_013503Escherichia coli4264443pMbo4.6NC_013500Moraxella bovis465839pMCHL01NC_011758Methylobacterium chloromethanicum CM438020766pMG160NC_004527Rhodobacter blasticus343167pMG828-2NC_008487Escherichia coli409150pMG828-4NC_008489Escherichia coli746248pMMCU1NC_013056Acinetobacter calcoaceticus877135pMMCU2NC_013506Acinetobacter baumannii1027036pMRAD01NC_010510Methylobacterium radiotolerans JCM 283158616470pMS260NC_005312Actinobacillus pleuropneumoniae812461pNGR234aNC_000914Rhizobiumsp. NGR23453616559Lrp4170197189196845Lrp3089188867188430pNGR234bNC_012586Rhizobiumsp. NGR234243003362Lrp4690656547656107Lrp4585667494667913Lrp439010380201038463Lrp4485682796683215Lrp389624008492401319Lrp4479709104708715Lrp41892833628761Lrp338911089001109337Lrp3690703213702764Lrp327711129531112582pNL2NC_009427Novosphingobium aromaticivorans DSM 1244448726866pO111_4NC_013367Escherichia coli O111:H- str. 11128814050pO26-S4NC_011228Escherichia coli675851pOLA52NC_010378Escherichia coli5160246pOU1114NC_010421Salmonella entericasubsp.entericaserovar Dublin3459542pOU1115NC_010422Salmonella entericasubsp.entericaserovar Dublin7458949pPNC_003455Salmonella entericasubsp.entericaserovarEnteritidis430150pP742405NC_011733Cyanothecesp. PCC 74241808338pP742406NC_011734Cyanothecesp. PCC 74241521940pPMA4326CNC_005921Pseudomonas syringaepv.maculicola824453pPNAP07NC_008763Polaromonas naphthalenivorans CJ2989857pPRO2NC_008608Pelobacter propionicus DSM 23793072256pPT1NC_002143Comamonas testosteroni1539856pR132501NC_012848Rhizobium leguminosarumbv. trifolii WSM132582892460Lrp4788234905234471Lrp4486386338386760Lrp3993645542645087Lrp4279147165146776pR132502NC_012858Rhizobium leguminosarumbv. trifolii WSM132566097361pR132503NC_012853Rhizobium leguminosarumbv. trifolii WSM132551608859HU4794300662300916pR132504NC_012852Rhizobium leguminosarumbv. trifolii WSM132535031261pR132505NC_012854Rhizobium leguminosarumbv. trifolii WSM132529478260pRFNC_007110Rickettsia felis URRWXCal26282934pRFdeltaNC_007111Rickettsia felis URRWXCal23926333pRi1724NC_002575Agrobacterium rhizogenes21759457pRi2659NC_010841Agrobacterium rhizogenes18546258pRL10NC_008381Rhizobium leguminosarumbv. viciae 384148813560pRL11NC_008384Rhizobium leguminosarumbv. viciae 384168420261pRL12NC_008378Rhizobium leguminosarumbv. viciae 384187002161Lrp4688599116598682Lrp4388658287658718Lrp39934560145146Lrp4279450080449691pRL7NC_008382Rhizobium leguminosarumbv. viciae 384115156458HU48942048420230pRL8NC_008383Rhizobium leguminosarumbv. viciae 384114746359Lrp33877076370344pRLG201NC_011368Rhizobium leguminosarumbv. trifolii WSM2304126610560Lrp4589917573917136Lrp44854199842417Lrp4479473039472650Lrp409311621461161691Lrp409311509391150484Lrp3288707587707162pRMNC_010927Rickettsia monacensis2348632pSC101NC_002056Salmonella entericasubsp.entericaserovar Typhimurium926351pSE11-6NC_011411Escherichia coli SE11408249pSE34NC_010860Salmonella entericasubsp.entericaserovar Enteritidis3295041pSMED01NC_009620Sinorhizobium medicae WSM419157095162Lrp4077143180143557Lrp3489574284573847pSMED02NC_009621Sinorhizobium medicae WSM419124540860Lrp4291556486556932Lrp4091842324842758Lrp31872234521917pSmeSM11aNC_013545Sinorhizobium meliloti14417060Lrp46967044970922pSmeSM11bNC_010865Sinorhizobium meliloti SM1118125159pSMS35_4NC_010486Escherichia coli SMS-3-5407450pSx-QyyNC_006826Sphingobium xenophagum568356pSymANC_003037Sinorhizobium meliloti 1021135422660LrppSymBNC_003078Sinorhizobium meliloti 1021168333362Lrp3890440778440335Lrp36892955529992pTB3NC_008388Roseobacter denitrificans OCh 1141657555pTcM1NC_010600Acidithiobacillus caldus6515857IHF56892518625449pTiS4NC_011982Agrobacterium vitis S425882457HU41942735627102HU40948340883154Lrp42799692097309pTi-SAKURANC_002147Agrobacterium tumefaciens20647956HU44949576395509pUT1NC_014005Sphingobium japonicum UT26S3177664pUT2NC_014009Sphingobium japonicum UT26S539861pXAUT01NC_009717Xanthobacter autotrophicus Py231616465pXCV19NC_007505Xanthomonas campestrispv.vesicatoriastr. 85-101914660pXF51NC_002490Xylella fastidiosa 9a5c5115850pYAN-1NC_008246Sphingobium yanoikuyae518262pYAN-2NC_008247Sphingobium yanoikuyae492464RSF1010NC_001740Escherichia coli868461Symbiotic plasmid p42dNC_004041Rhizobium etli CFN 4237125458TiNC_002377Agrobacterium tumefaciens19414055IHF4397180164180436TiNC_003065Agrobacterium tumefaciensstr. C5821423357HU4494139735139481Ti plasmid pTiBo542NC_010929Agrobacterium tumefaciens24497855IHF3686209743209489IHF4598187204187479UnnamedNC_011143Phenylobacterium zucineum HLK138297669aThis list is the result of a TBLASTN analysis using the 300 N-terminal amino acid sequence of protein MobA_RSF1010 as a query under strict conditions (i.e., thresholds of 30% identity and 70% query coverage).bAverage G+C content of the plasmid.cReported TBLASTN identity to each NAP.


### 3.6. Conclusions

We compared the distribution of NAP gene homologs among plasmids and plasmid features. Larger plasmids frequently had NAP gene homologs, possibly to maintain themselves and host cell fitness. Plasmids with NAP gene homologs also frequently carried relaxase genes. Although this may be related to their relatively larger sizes, together with the fact that NAPs affect global gene regulation, it is likely that NAPs contribute to plasmid transmission. Considering the fact that NAPs encoded on plasmids actually help the host cell to integrate newly acquired genes into host regulatory networks [14, 15], large plasmids with NAP gene homologs may be generally more beneficial not only for the host cell, but also for their own existence.NAP homologs encoded on plasmids can interact with different types of NAPs encoded on the host chromosome and cooperatively regulate host transcriptional networks. Understanding these mechanisms in more detail will shed light on the meanings of the distributions of NAPs on plasmids and chromosomes. Comprehensive analysis of their binding sites in the host and plasmid genomes will help us to understand the relationships between G+C content and the presence of NAPs. Such information will explain how bacteria adapt and evolve by acquiring foreign genes by HGT.


## 3.1. Database Collection and Plasmid Classification by Origin

We downloaded the whole sequences of 2278 plasmids from the NCBI ftp site (April 2010). Duplicated plasmids were removed manually, and the resultant 2260 plasmid sequences were used in this study. To understand what types of plasmids were included in the database, we classified them into six groups according to their source organisms. The database included 1382 Gram-negative, 725 Gram-positive, 81 archaeal, 43 eukaryotic, 1 viral, and 28 unclassified plasmids.


## 3.2. Identification of the Plasmids Containing NAP Gene Homologs

Using the amino acid sequences of well-known NAPs (Fis, H-NS, HU, IHF, and Lrp) and newly found NAPs (MvaT and NdpA), their distributions were surveyed for plasmids using the TBLASTN program. Some plasmids had ORFs showing sequence similarities to both HU and IHF. We adopted the one with the higher E value. Of 2260 plasmids, 155 (7%) contained the gene encoding NAP homolog. Of those, 116 (75%) contained only one NAP gene homolog and 39 (25%) contained more than one NAP gene homolog. No plasmids carried the Fis gene homolog. Twenty-two plasmids carried the H-NS gene homolog, and all of them had a Gram-negative origin (Table1). Sixty-six plasmids had the HU gene homolog; of these, 51 had a Gram-negative origin and 15 had a Gram-positive origin (Table 2). Twenty-seven plasmids (25 with Gram-negative and 2 with Gram-positive origins) carried the IHF gene homolog (Table 3). Forty-eight plasmids (46 with Gram-negative, 1 with a Gram-positive, and 1 with an archaeal origin) carried the Lrp gene homolog (Table 4). Of these, 23 (48%) contained more than one Lrp gene homolog. On the other hand, MvaT and NdpA homologs were encoded on only 3 plasmids, and all of them were of Gram-negative origin (Table 5). Previously reported plasmids that are known to have NAP gene homologs were included in those 155 plasmids. These included R27 (NC_002305) and pHCM1 (NC_003384) [18, 19] with the H-NS gene homolog; pQBR103 (NC_009444) [20] with the HU and NdpA gene homologs; and pCAR1 (NC_004444) [21, 22] with the MvaT, HU, and NdpA gene homologs. These results indicated the adequacy of our search. Because we used NAPs from Gram-negative bacteria as query sequences, it may be reasonable that 136 (88%) of 155 plasmids with the NAP gene homolog belonged to the group isolated from Gram-negative bacteria. Therefore, in further studies we discussed the Gram-negative plasmid group.Table 1
Plasmids containing the gene encoding H-NS homologa.
Plasmid nameAccession no.Source organismLength (nt)G+C content (%)bIdentity (%)cQuery coverage (%)Subject startSubject endClassificationdMOB familye1NC_013972Erwinia amylovora ATCC 499462824350669931292728−pAsa5NC_009350Aeromonas salmonicidasubsp.salmonicida A449155098544699941534−MOBF47991689016483pAsal5NC_009352Aeromonas salmonicidasubsp.salmonicida185365446991228512692−pEA29NC_013957Erwinia amylovora CFBP14302825950669931292728−pEA29NC_005706Erwinia amylovora2818550649929912590−pEC-IMPNC_012555Enterobacter cloacae318782486499109370108969−MOBHpEC-IMPQNC_012556Enterobacter cloacae324503486499109370108969−MOBHpEJ30NC_004834Erwiniasp. Ejp 5562959350669946514250−pEP36NC_013263Erwinia pyrifoliae Ep1/96359095066992504025441−pEP36NC_004445Erwinia pyrifoliae Ep1/963590450649846754280−pET45NC_010699Erwinia tasmaniensis Et1/99446945152933743537809−MOBFpET49NC_010697Erwinia tasmaniensis Et1/99487514436943082131204−pHCM1NC_003384Salmonella entericasubsp.entericaserovar Typhi str. CT18218160486199131861131460−MOBHpK2044NC_006625Klebsiella pneumoniae NTUH-K20442241525067993571736112−plasmid_153kbNC_009705Yersinia pseudotuberculosis IP 317581531404044100139846140265−pLVPKNC_005249Klebsiella pneumoniae219385506799114397114792−pMAK1NC_009981Salmonella entericasubsp.entericaserovar Choleraesuis2084094761996004659645−MOBHpO111_1NC_013365Escherichia coli O111:H- str. 111282046044761998017579774−MOBHpSG1NC_007713Sodalis glossinidiusstr. “morsitans”8330649439725332922−R27NC_002305Salmonella entericasubsp.entericaserovar Typhi180461466199148225148626−MOBHR478NC_005211Serratia marcescens274762466499111747111346−MOBHUnnamedNC_011148Salmonella entericasubsp.entericaserovar Agona str. SL4833797841439576717288−aThis list is the result of a TBLASTN analysis using the amino acid sequence of H-NS as a query under strict conditions (i.e., thresholds of 30% identity and 70% query coverage). Besides these plasmids, pSf-R27 from Shigella flexneri2a str. 2457T was completely sequenced by Wei et al. [17] and encodes the H-NS-like protein Sfh.bAverage G+C content of the plasmid.cReported TBLASTN identity to H-NS.dPlasmid classification according to its source organism (−, Gram-negative plasmid).ePlasmid classification according to its relaxase gene sequence as described by Garcillán-Barcia et al. [16].Table 2
Plasmids containing the gene encoding HU homologa.
Plasmid nameAccession no.Source organismLength (nt)G+C content (%)bIdentity (%)cQuery coverage (%)Subject startSubject endClassificationdMOB familye1NC_006823Aromatoleum aromaticum EbN1207355585599186175185909−1NC_007949Polaromonassp. JS6663604055752996105260786−MOBH1NC_008010Deinococcus geothermalis DSM 11300574127663897550805550545+1NC_008503Lactococcus lactissubsp.cremoris SK1114041343794973210007+MOBP1NC_008242Chelativoranssp. BNC1343931624194133932133678−MOBQ2NC_012529Deinococcus deserti VCD115314317643893269648269899+3NC_012528Deinococcus deserti VCD11539645961409687008957+MegaplasmidNC_007974Cupriavidus metallidurans CH34258008464519913934151393149−MOBVMegaplasmidNC_005863Desulfovibrio vulgarisstr.Hildenborough20230166319855025765−Megaplasmid pDF308NC_013940Deferribacter desulfuricans SSM13085442441100253817253548−Megaplasmid pHG1NC_005241Ralstonia eutropha H16452156624899343060342791−p49879.1NC_006907Leptospirillum ferrooxidans2887858479932813015−MOBQp49879.2NC_006909Leptospirillum ferrooxidans280125548991585815592−MOBQpAH187_270NC_011655Bacillus cereus AH1872700823459100113139112870+pAH820_272NC_011777Bacillus cereus AH8202721453458100153060152791+pAM04528NC_012693Salmonella enterica1582135257991406714333−MOBHpAOVO01NC_008765Acidovoraxsp. JS427268962461006514064871−MOBFpAPA01-011NC_013210Acetobacter pasteurianus IFO 3283-011917995347100154736154467−46993844238708pAR060302NC_012692Escherichia coli1665305357991575516021−MOBHpAsa4NC_009349Aeromonas salmonicidasubsp.salmonicida A4491667495360992684426578−MOBHpAtS4cNC_011984Agrobacterium vitis S4211620594594141245140991−MOBQpAtS4eNC_011981Agrobacterium vitis S46317755741944047640222−MOBQpBc239NC_011973Bacillus cereus Q12392463352100191895192164+pBF9343NC_006873Bacteroides fragilis NCTC 9343365603235921580315558−MOBPpBPHY01NC_010625Burkholderia phymatum STM8151904893624399826527826252−pBPHY02NC_010627Burkholderia phymatum STM8155951085945999862598359−pBtoxisNC_010076Bacillus thuringiensisserovar israelensis1279233252997738277648+pBWB401NC_010180Bacillus weihenstephanensis KBAB44170543459100338347338078+pCAR1NC_004444Pseudomonas resinovorans1990355642999780998075−MOBHpCAUL01NC_010335Caulobactersp. K312336496744999759897329−MOBQpCER270NC_010924Bacillus cereus2700823459100169548169279+pDBORONC_009137Lactococcus lactissubsp.lactisbv. diacetylactis164043537941638716112+pDVUL01NC_008741Desulfovibrio vulgaris DP4198504663198198317198054−peH4HNC_012690Escherichia coli1481055357991406714333−MOBHpG9842_209NC_011775Bacillus cereus G984220948830601008882888559+pH308197_258NC_011339Bacillus cereus H3081.9725848434591008303383302+pHD5ATNC_012752Candidatus Hamiltonella defensa 5AT (Acyrthosiphon pisum)590324545991498115247−MOBPpIP1202NC_009141Yersinia pestisbv. Orientalis str. IP2751829135357991406714333−MOBHplasmid 2NC_007972Cupriavidus metallidurans CH34171459614699125530125261−pMOL28NC_006525Cupriavidus metallidurans CH341714616146995152951798−pMP118NC_007930Lactobacillus salivarius UCC1182424363254995676356497+MOBVpNPUN02NC_010632Nostoc punctiforme PCC 731022549184144997480474538−MOBVpOANT02NC_009670Ochrobactrum anthropi ATCC 491881014915949943270032446−pP91278NC_008613Photobacterium damselaesubsp.piscicida131520525799125918126184−MOBHpP99-018NC_008612Photobacterium damselaesubsp.piscicida150157515799133314133580−MOBHpPER272NC_010921Bacillus cereus2721453458100153060152791+pPMA4326ANC_005918Pseudomonas syringaepv.maculicola4669755429915201786−pPMA4326BNC_005919Pseudomonas syringaepv.maculicola4011055459914571723−pQBR103NC_009444Pseudomonas fluorescens SBW25425094535199182862183128−pR132503NC_012853Rhizobium leguminosarumbv. trifolii WSM1325516088594794300662300916−MOBQpRA1NC_012885Aeromonas hydrophila1439635158991557315839−MOBHpRALTANC_010529Cupriavidus taiwanensis557200604698153542153276−pREB1NC_009926Acaryochloris marina MBIC110173741614746100339743340012−MOBFpREB2NC_009927Acaryochloris marina MBIC1101735608745481005758357852−MOBFpREB3NC_009928Acaryochloris marina MBIC110172731214546100234682234951−MOBF42100243339243608pRL7NC_008382Rhizobium leguminosarumbv. viciae 38411515645848942048420230−MOBQpRLG203NC_011370Rhizobium leguminosarumbv. trifolii WSM2304308747584994141121140867−pRp12D01NC_012855Ralstonia pickettii 12D389779583799321346321080−MOBHpSG2NC_007184Sodalis glossinidius27240454586100729845−pSG3NC_007186Sodalis glossinidius1920151511001381213543−pSN254NC_009140Salmonella entericasubsp.entericaserovar Newport str. SL2541764735357991406714333−MOBHpTiS4NC_011982Agrobacterium vitis S42588245741942735627102−MOBQ40948340883154pTi-SAKURANC_002147Agrobacterium tumefaciens2064795644949576395509−MOBQpVSAL840NC_011311Aliivibrio salmonicida LFI1238835404060993136131627−MOBF58997735077084pYR1NC_009139Yersinia ruckeri1580385157991507015336−MOBHTiNC_003065Agrobacterium tumefaciens str. C58214233574494139735139481−MOBQaThis list is the result of a TBLASTN analysis using the amino acid sequence of HUα or HUβ as a query under strict conditions (i.e., thresholds of 30% identity and 70% query coverage).bAverage G+C content of the plasmid.cReported TBLASTN identity to HU.dPlasmid classification according to its source organism (−, Gram-negative plasmid; +, Gram-positive plasmid).ePlasmid classification according to its relaxase gene sequence as described by Garcillán-Barcia et al. [16].Table 3
Plasmids containing the gene encoding IHF homologa.
Plasmid nameAccession no.Source organismLength (nt)G+C content (%)bIdentity (%)cQuery coverage (%)Subject startSubject endClassificationdMOB familyeAtNC_003064Agrobacterium tumefaciensstr. C58542868573682112654112412−MOBQMegaplasmidNC_012811Methylobacterium extorquens AM11261460683394720582720860−p2META1NC_012809Methylobacterium extorquens AM1378586544952836928635−MOBQpAACI01NC_013206Alicyclobacillus acidocaldarius subsp.acidocaldarius DSM 446917265443806266862432+pACHL01NC_011879Arthrobacter chlorophenolicus A6426858643292408818408546+pALVIN02NC_013862Allochromatium vinosum DSM 180399295360981090210627−pAph01NC_013193Candidatus Accumulibacter phosphatis clade IIAstr. UW-1167595625695144197144463−MOBPpAph03NC_013191Candidatus Accumulibacter phosphatis clade IIAstr. UW-13769559589754125140−pAtK84bNC_011990Agrobacterium radiobacter K841846685938865410953855−MOBQpAtK84cNC_011987Agrobacterium radiobacter K84388169574393340807340532−46931032710052pAtS4bNC_011991Agrobacterium vitis S41304355647974488045152−MOBQpBBta01NC_009475Bradyrhizobiumsp. BTAi122882661398666426388−pBFY46NC_006297Bacteroides fragilis YCH46337163435892509825343−MOBPpBIND01NC_010580Beijerinckia indicasubsp.indica ATCC 9039181736563677179816179601−MOBFpCHQ1NC_014007Sphingobium japonicum UT26S1909746336906311163377−pGLOV01NC_010815Geobacter lovleyi SZ771135338924119641468−pM44601NC_010373Methylobacteriumsp. 4-465795165359778067534−pMPOP01NC_010727Methylobacterium populi BJ001251646549931063510375−pMRAD03NC_010514Methylobacterium radiotolerans JCM 2831429856338942677826515−MOBFpMRAD04NC_010517Methylobacterium radiotolerans JCM 2831377436438941076310500−pPRO1NC_008607Pelobacter propionicus DSM 2379202397484194129679129957−pRSPA01NC_009429Rhodobacter sphaeroides ATCC 17025877879684997783519783791−pSWIT01NC_009507Sphingomonas wittichii RW1310228644095106554106820−MOBF36923534135069pTcM1NC_010600Acidithiobacillus caldus651585756892518625449−MOBP, MOBQpXCV183NC_007507Xanthomonas campestrispv. vesicatoriastr. 85-10182572603395138753138490−TiNC_002377Agrobacterium tumefaciens194140554397180164180436−MOBQTi plasmid pTiBo542NC_010929Agrobacterium tumefaciens244978553686209743209489−MOBQ4598187204187479aThis list is the result of a TBLASTN analysis using the amino acid sequence of IHFα or IHFβ as a query under strict conditions (i.e., thresholds of 30% identity and 70% query coverage).bAverage G+C content of the plasmid.cReported TBLASTN identity to IHF.dPlasmid classification according to its source organism (−, Gram-negative plasmid; +, Gram-positive plasmid).ePlasmid classification according to its relaxase gene sequence as described by Garcillán-Barcia et al. [16].Table 4
Plasmids containing the gene encoding Lrp homologa.
Plasmid nameAccession no.Source organismLength (nt)G+C content (%)bIdentity (%)cQuery coverage (%)Subject startSubject endClassificationdMOB familye1NC_008688Paracoccus denitrificans PD1222653815674192252075251623−429346421846467336966393416398113785110140109724ANC_009007Rhodobacter sphaeroides 2.4.11140456939933024129789−MOBFBNC_007488Rhodobacter sphaeroides 2.4.11141787043968186181385−bglu_1pNC_012723Burkholderia glumae BGR1133591613690124017123577−MegaplasmidNC_008043Ruegeriasp. TM1040821788594184143820144233−41916872576877063691734136733690MegaplasmidNC_007974Cupriavidus metallidurans CH34258008464448811712451170814−MOBV409111697021169256389715867261586250MegaplasmidNC_006569Ruegeria pomeroyi DSS-3491611633688356303355869−MOBCMegaplasmidNC_007336Ralstonia eutropha JMP134634917613593377503377045−p42eNC_007765Rhizobium etli CFN 42505334623471255037255384−p42fNC_007766Rhizobium etli CFN 42642517614588436907437341−4391406350405901418549138349179939952106342110983996199426199899pAB510aNC_013855Azospirillumsp. B5101455109685788274908275342−4495979549980013329411803351179874pAB510bNC_013856Azospirillumsp. B510723779674484471830472243−3294318139318600pAB510cNC_013857Azospirillumsp. B510681723674585408064407645−34913638536834pAB510dNC_013858Azospirillumsp. B510628837684479472768472379−409032318432274137872814382818663085619027618623pAtS4eNC_011981Agrobacterium vitis S4631775573087460443460871−MOBQ3474425247424888pBPHY01NC_010625Burkholderia phymatum STM815190489362468511536081154027−pBPHY02NC_010627Burkholderia phymatum STM815595108594191271795271346−pCNC_010997Rhizobium etli CIAT 6521091523614688617696618130−MOBQ4290609059608619399541773841820242797148047151933993406570407025pCAUL01NC_010335Caulobactersp. K31233649673489182479182042−MOBQpEST4011NC_005793Achromobacter denitrificans769586258884122440793−MOBP58883423333802pGMI1000MPNC_003296Ralstonia solanacearum GMI1000209450967439817379581738437−4693822030821572pHV4NC_013966Haloferax volcanii DS2635786623371401763401410ArchaeapIJB1NC_013666Burkholderia cepacia994486358887490775338−MOBPpK2044NC_006625Klebsiella pneumoniae NTUH-K2044224152503390194643195086−pLVPKNC_005249Klebsiella pneumoniae2193855033904623646679−pMLaNC_002679Mesorhizobium loti MAFF303099351911593293185603185148−3089207314206877pMLbNC_002682Mesorhizobium loti MAFF3030992083156037932463224177−pNGR234aNC_000914Rhizobiumsp. NGR234536165584170197189196845−MOBQ3089188867188430pNGR234bNC_012586Rhizobiumsp. NGR2342430033624690656547656107−MOBQ458566749466791343901038020103846344856827966832153896240084924013194479709104708715418928336287613389110890011093373690703213702764327711129531112582pPNAP04NC_008760Polaromonas naphthalenivorans CJ2143747593590142511142068−pR132501NC_012848Rhizobium leguminosarumbv.trifolii WSM1325828924604788234905234471−MOBQ448638633838676039936455426450874279147165146776pRALTANC_010529Cupriavidus taiwanensis557200603891465839465393−pRHL1NC_008269Rhodococcus jostii RHA11123075653691854207854656+3384783666783253pRL12NC_008378Rhizobium leguminosarumbv. viciae 3841870021614688599116598682−MOBQ4388658287658718399345601451464279450080449691pRL8NC_008383Rhizobium leguminosarumbv. viciae 38411474635933877076370344−MOBQpRLG201NC_011368Rhizobium leguminosarumbv. trifolii WSM23041266105604589917573917136−MOBQ4485419984241744794730394726504093116214611616914093115093911504843288707587707162pRSKD131ANC_011962Rhodobacter sphaeroides KD131157345704296148295147819−pRSKD131BNC_011960Rhodobacter sphaeroides KD1311033557039939840097948−pRSPA01NC_009429Rhodobacter sphaeroides ATCC 170258778796840903130930866−3988659383658952pRSPH01NC_009040Rhodobacter sphaeroides ATCC 17029122606703993118088118540−pSMED01NC_009620Sinorhizobium medicae WSM4191570951614077143180143557−MOBQ3489574284573847pSMED02NC_009621Sinorhizobium medicae WSM4191245408604291556486556932−MOBQ409184232484275831872234521917pSMED03NC_009622Sinorhizobium medicae WSM419219313604695105044105508−pSmeSM11aNC_013545Sinorhizobium meliloti1441706046967044970922−MOBQpSymANC_003037Sinorhizobium meliloti 1021135422660438910606991060262−MOBQpSymBNC_003078Sinorhizobium meliloti 10211683333623890440778440335−MOBQ36892955529992pTiS4NC_011982Agrobacterium vitis S42588245742799692097309−MOBQUnnamedNC_011961Thermomicrobium roseum DSM 5159917738663085736739737146−MOBPaThis list is the result of a TBLASTN analysis using the amino acid sequence of Lrp as a query under strict conditions (i.e., thresholds of 30% identity and 70% query coverage).bAverage G+C content of the plasmid.cReported TBLASTN identity to Lrp.dPlasmid classification according to its source organism (−, Gram-negative plasmid; +, Gram-positive plasmid).ePlasmid classification according to its relaxase gene sequence as described by Garcillán-Barcia et al. [16].Table 5
Plasmids containing the gene encoding MvaT or NdpA homologa.
Plasmid nameAccession no.Source organismLength (nt)G+C content (%)bIdentity (%)cQuery coverage (%)Subject startSubject endClassificationdMOB familyeMvaTpCAR1NC_004444Pseudomonas resinovorans1990355661987764077993−MOBHpQBR103NC_009444Pseudomonas fluorescens SBW254250945361969807697717−pWW53NC_008275Pseudomonas putida10792957619884158768−NdpAp0908NC_010113Vibriosp. 0908814134951997973178736−pCAR1NC_004444Pseudomonas resinovorans1990355636989539094395−MOBHpQBR103NC_009444Pseudomonas fluorescens SBW25425094533199161413160400−aThis list is the result of a TBLASTN analysis using the amino acid sequence of MvaT or NdpA as a query under strict conditions (i.e., thresholds of 30% identity and 70% query coverage).bAverage G+C content of the plasmid.cReported TBLASTN identity to MvaT or NdpA.dPlasmid classification according to its source organism (−, Gram-negative plasmid).ePlasmid classification according to its relaxase gene sequence as described by Garcillán-Barcia et al. [16].


## 3.3. Relationships between Plasmid Size and NAP Gene Homolog Distributions

We first compared the sizes of 136 plasmids with NAP gene homologs with those of all 1382 Gram-negative group plasmids. All 1382 plasmids could be divided into 4 groups according to size, small (<10 kb), intermediate (10 to 100 kb), large (100 kb to 1 Mb), and mega (>1 Mb) plasmids. The distribution of the 136 plasmids, each of which had one or more genes encoding NAP homologs, is shown in Figure1(a): none of 415 small plasmids, 34 (5%) of 686 intermediate plasmids, 90 (33%) of 269 large plasmids, and 12 (100%) of 12 mega plasmids carried at least one NAP gene homolog. The average size of the 136 plasmids was larger (364 kb) than that of all 1382 plasmids (83 kb). These results suggest that larger plasmids, especially >100 kb, frequently have NAP gene homologs. Carrying large plasmids may reduce host fitness more than carrying small plasmids because the former have more genes that can disrupt transcriptional networks in the host cell. In addition, large plasmids may have more binding sites for NAPs than small plasmids. Because chromosome-encoded NAPs bind to both chromosomes and plasmids, carrying large plasmids may also result in a reduction in the binding of NAPs to the host chromosome, causing undesirable effects on the host cell. Plasmid-encoded NAP homologs may interact with chromosome-encoded NAPs, coordinately sustain the structure of both chromosome and plasmid, and regulate the transcriptional regulation network [23]. In fact, recent studies have shown that some plasmid-encoded NAP homologs can complement the depletion of chromosomal NAPs and optimize gene transcription both on plasmids and in the host chromosome [14, 15, 24]. Thus, larger plasmids may have NAP gene homologs to maintain host cell fitness. In addition, the average size of the 38 plasmids containing more than one NAP gene homolog was larger (790 kb) than that of the 98 plasmids containing only one NAP gene homolog (199 kb). This suggests that particularly large plasmids have many NAP gene homologs to maintain themselves in the host cell.Size comparison of the Gram-negative plasmids with and without NAP gene homologs. (a) A total of 136 Gram-negative plasmids with one or more NAP gene homologs and 1246 Gram-negative plasmids without NAP gene homologs are shown by black and white bars, respectively. (b) Gram-negative plasmids with each NAP gene homolog are as follows: H-NS, red; HU, blue; IHF, green; Lrp, purple; MvaT, yellow; and NdpA, orange.
(a)(b)Distributions of the NAP genes on proteobacterial genomes were also surveyed using the TBLASTN program. The average size of the completely sequenced bacterial genomes was 3.25 Mb and 1054 NAP genes (100, Fis; 125, H-NS; 236, HU; 247, IHF; 127, Lrp; 119, MvaT; and 100, NdpA) were found in 588 proteobacterial genomes. Frequency of NAP genes in plasmids was higher (1 per 236 kb) than that in proteobacterial genomes (1 per 1.8 Mb), also suggesting that larger plasmids frequently have NAP gene homologs to minimize their negative effects on the host cell.Of the plasmids with the NAP gene homolog, the average size of those with the H-NS gene homolog was relatively small (132 kb) while that of those with the Lrp gene homolog was relatively large (725 kb). The average sizes of those with the other NAP gene homologs were as follows: HU (301 kb), IHF (230 kb), MvaT (244 kb), and NdpA (235 kb) (Figure1(b)). H-NS exists in an oligomeric form and binds to DNA, especially A+T-rich regions, by bridging it [25]. This function may be important for regulating gene expression on relatively small plasmids among those with the NAP gene homolog. The activity of H-NS can also be modulated by Hha-like proteins [26]. Intriguingly, TBLASTN analysis showed that 12 (55%) of 22 plasmids with the H-NS gene homolog also carried gene encoding Hha-like protein although only 65 (5%) of all 1382 plasmids carried Hha-like protein gene (Table 6). This suggests the close relationship of H-NS and Hha-like protein. On the other hand, Lrp exists in dimeric, octameric, and hexadecameric forms and compacts DNA by wrapping it [27]. This distinctive DNA-binding ability may be essential for maintaining the structure of particularly larger plasmids.Table 6
Gram-negative plasmids containing the gene encoding Hha-like proteina.
Plasmid nameAccession no.Source organismLength (nt)G+C content (%)bNAP gene homologIdentity (%)cQuery coverage (%)Subject startSubject endMOB familyd55989pNC_011752Escherichia coli 5598972482465392100259828NR1NC_009133Escherichia coli942895253928719387390MOBFp1658/97NC_004998Escherichia coli1254915155923641936616MOBFp1ESCUMNC_011749Escherichia coli UMN0261223015053925350853311MOBFp2ESCUMNC_011739Escherichia coli UMN0263380942629076827488MOBQp53638_226NC_010719Escherichia coli 536382256834855926761567418MOBFpAPEC-O1-RNC_009838Escherichia coli APEC O12413874650926138961586MOBHpAPEC-O2-ColVNC_007675Escherichia coli18450149559238823685MOBFpAPEC-O2-RNC_006671Escherichia coli10137553539248564659MOBFpBS512_211NC_010660Shigella boydii CDC 3083-94210919465589190719190910MOBFpBS512_33NC_010657Shigella boydii CDC 3083-943310341629028943088pC15-1aNC_005327Escherichia coli923535353928749087687MOBFpCP301NC_004851Shigella flexneri2a str. 301221618465592207828208025MOBFpCROD1NC_013717Citrobacter rodentium ICC168544494756925322053417pCROD2NC_013718Citrobacter rodentium ICC168392654262901552615332pCT02021853_74NC_011204Salmonella entericasubsp.entericaserovar Dublin str. CT_02021853745514962904848248288MOBQpCTX-M3NC_004464Citrobacter freundii894685138712613626294MOBP8946831964064840439pCTXM360NC_011641Klebsiella pneumoniae680185138716455164709MOBP6801831961092710718pCVM29188_146NC_011076Salmonella entericasubsp.entericaserovar Kentucky str. CVM291881468114953921875518558MOBFpEC14_114NC_013175Escherichia coli114222515392113985114182MOBFpEC-IMPNC_012555Enterobacter cloacae31878248H-NS50926049160688MOBHpEC-IMPQNC_012556Enterobacter cloacae32450348H-NS50926049160688MOBHpEG356NC_013727Shigella sonnei702755253926944469641MOBFpEK499NC_013122Escherichia coli1175365353924198542182pEK516NC_013121Escherichia coli644715353923141031213pEL60NC_005246Erwinia amylovora601455138712318723345MOBP6014531963786337654pEntH10407NC_013507Escherichia coli ETEC H10407670945155784342143254MOBFpHCM1NC_003384Salmonella entericasubsp.entericaserovar Typhi str. CT1821816048H-NS47100105911106117MOBHpK2044NC_006625Klebsiella pneumoniae NTUH-K204422415250H-NS, Lrp4585143331143528pK29NC_010870Klebsiella pneumoniae2696744650925932259519MOBHpKF3-70NC_013542Klebsiella pneumoniae700575253921596715770MOBFpKF3-94NC_013950Klebsiella pneumoniae9421952589695969390MOBFpKP187NC_011282Klebsiella pneumoniae 342187922476496110083109877187922428915501344pKPN3NC_009649Klebsiella pneumoniaesubsp.pneumoniae MGH 785781758795259975693056721MOBFplasmid_153 kbNC_009705Yersinia pseudotuberculosis IP 3175815314040H-NS6993633426354215314056924973449931pLVPKNC_005249Klebsiella pneumoniae21938550H-NS, Lrp61971480561478472193854585214828215025pMAK1NC_009981Salmonella entericasubsp.entericaserovar Choleraesuis20840947H-NS471004920849414MOBHpMAS2027NC_013503Escherichia coli426444362901968519491MOBQpO103NC_013354Escherichia coli O103:H2 str. 12009755464955925172751924MOBFpO111_1NC_013365Escherichia coli O111:H- str. 1112820460447H-NS471006692567131MOBHpO111_3NC_013366Escherichia coli O111:H- str. 11128776905055921197512172MOBFpO157NC_013010Escherichia coli O157:H7 str. TW14359946014855927079270989pO157NC_011350Escherichia coli O157:H7 str. EC4115946444855925473554932pO157NC_007414Escherichia coli O157:H7 EDL9339207748559216671864pO157NC_002128Escherichia coli O157:H7 str. Sakai927214855927118371380pO26INC_011812Escherichia coli729465153926660866805MOBFpO86A1NC_008460Escherichia coli120730495592101598101795MOBFpOLA52NC_010378Escherichia coli516024662901211411920MOBQpOU1114NC_010421Salmonella entericasubsp. entericaserovar Dublin3459541629054465252MOBQpOU1115NC_010422Salmonella entericasubsp.entericaserovar Dublin745894962903724637052MOBQpSB4_227NC_007608Shigella boydii Sb227126697475592110688110885MOBFpSE11-1NC_011419Escherichia coli SE111000215056925840758210MOBPpSE34NC_010860Salmonella entericasubsp.entericaserovar Enteritidis329504162902187522069MOBQpSFO157NC_009602Escherichia coli12123950527517091870MOBFpSG1NC_007713Sodalis glossinidiusstr. “morsitans”8330649H-NS489222942491pSG1NC_007182Sodalis glossinidius815534948925621756414pSMS35_130NC_010488Escherichia coli SMS-3-513044051559233643167MOBFpSS_046NC_007385Shigella sonnei Ss046214396455592178363178560MOBFpUTI89NC_007941Escherichia coli UTI89114230515392113993114190MOBFpWR501NC_002698Shigella flexneri221851465592207534207731MOBFR100NC_002134Shigella flexneri2b str. 222942815253928718587382MOBFR27NC_002305Salmonella entericasubsp.entericaserovar Typhi18046146H-NS47100159402159196MOBHR478NC_005211Serratia marcescens27476246H-NS50925942659623MOBHR721NC_002525Escherichia coli755824366903528535091UnnamedNC_011148Salmonella entericasubsp.entericaserovar Agona str. SL4833797841H-NS429313631163aThis list is the result of a TBLASTN analysis using the amino acid sequence of Hha as a query under strict conditions (i.e., thresholds of 30% identity and 70% query coverage).bAverage G+C content of the plasmid.cReported TBLASTN identity to Hha.dPlasmid classification according to its relaxase gene sequence as described by Garcillán-Barcia et al. [16].


## 3.4. Relationships between Plasmid G+C Content and NAP Gene Homolog Distributions

Next, we surveyed the G+C content of the Gram-negative group plasmids with and without NAP gene homologs. The average G+C content of the 136 plasmids with NAP gene homologs was higher (56.4%) than that of all 1382 plasmids (44.8%) (Figure2(a)). Note that the average G+C content of large and mega plasmids (55.0% and 62.9%, resp.) was higher than that of small and intermediate plasmids (44.8% and 40.4%). Considering that larger plasmids frequently had NAP gene homologs, this seems reasonable. Nevertheless, plasmids with H-NS gene homologs had a lower G+C content (48.3%) than did those with other NAP gene homologs, including HU (54.2%), IHF (58.7%), Lrp (62.3%), MvaT (55.6%), and NdpA (52.9%) (Figure 2(b)). H-NS family protein binds A+T-rich regions not only on chromosomes but also on plasmids [15]. Acquisition of a large A+T-rich plasmid with many H-NS binding sites may result in a reduction in the binding of H-NS to the host chromosome and host cell fitness [14]. It is therefore possible that large A+T-rich plasmids may have to supply another H-NS encoded on themselves to minimize the effect on the host cell. On the other hand, although MvaT-family proteins are the functional homolog of H-NS [10, 15], plasmids containing the MvaT gene homolog were not particularly low in G+C content. Although only three plasmids contained the MvaT gene homolog and thus we cannot discuss this interesting phenomenon in detail, the difference between H-NS and MvaT may be derived from their different origin or host bacteria.G+C content comparison of the Gram-negative plasmids with and without NAP gene homologs. (a) A total of 136 Gram-negative plasmids with one or more NAP gene homologs and 1246 Gram-negative plasmids without NAP gene homologs are shown by black and white bars, respectively. (b) Gram-negative plasmids with each NAP gene homolog are as follows: H-NS, red; HU, blue; IHF, green; Lrp, purple; MvaT, yellow; and NdpA, orange.
(a)(b)


## 3.5. Relationships between Plasmid Transferability and NAP Gene Homolog Distributions

Conjugative transfer is an essential function of plasmids, through which they play an important role in bacterial evolution and host cell behavior [11, 12]. Relaxase is an essential protein for plasmid transmission involved in the cleavage of the transferring DNA at the origin of transfer (oriT) site, and plasmids with relaxase genes are thought to be transmissible. Garcillán-Barcia et al. [16] proposed that transmissible plasmids can be classified into 6 MOB families (MOBC, MOBF, MOBH, MOBP, MOBQ, and MOBV) according to the amino acid sequences of 6 prototype relaxase proteins. MOBF and MOBH families are predominantly composed of conjugative plasmids, also called self-transmissible plasmids, and the other 4 families are composed of both mobilizable and conjugative plasmids. Recent studies have reported that plasmid-encoded H-NS family proteins have a “stealth” function and aide horizontal transfer of plasmids [14, 15]. Other NAPs also act as global transcriptional regulators and may regulate expression of genes involved in plasmid transmission. To discuss the relationship between NAP gene homolog distribution and plasmid transferability, we determined the distribution of genes encoding relaxase proteins in Gram-negative plasmids according to the classification by Garcillán-Barcia et al. [16]. Four hundred and nine (30%) of 1382 Gram-negative plasmids carried relaxase genes, and 71 (17%) of those 409 plasmids carried NAP gene homologs. Note that 71 (52%) of 136 plasmids with NAP gene homologs carried relaxase genes. This indicates that plasmids with NAP gene homologs frequently carried the relaxase genes than did those without NAP gene homologs. This phenomenon may be related to the average size of the plasmids. That of the 409 plasmids with relaxase genes was relatively larger (145 kb) than that of all 1382 plasmids (83 kb), corresponding to the fact that larger plasmids frequently had NAP gene homologs.Four hundred and nine plasmids were classified into each MOB family (13, MOBC; 128, MOBF; 29, MOBH; 86, MOBP; 131, MOBQ; and 26, MOBV). Plasmid 1 (NC_008545) was classified into both the MOBC and MOBF families. In addition, the MOBP, MOBQ, and MOBV families were partially overlapped as described by Garcillán-Barcia et al. [16]. Seventy-one plasmids with NAP gene homologs were contained in each MOB family (1, MOBC; 11, MOBF; 20, MOBH; 8, MOBP; 30, MOBQ; and 2, MOBV). Intriguingly, 20 (69%) of 29 MOBH-family plasmids encoded some NAP homologs, and most of them were H-NS or HU (Table 7). The MOBH family was composed of predominantly large conjugative plasmids, such as the IncHI1 group of plasmids, suggesting that HU may also contribute to plasmid transmission as does H-NS. Furthermore, 30 (23%) of 131 MOBQ-family plasmids also contained some NAP gene homologs, and 15 (50%) of those carried Lrp gene homologs (Table 8). The MOBQ family was composed of both mobilizable and conjugative plasmids, such as those of Rhizobium and Agrobacterium, implying that Lrp may also affect plasmid conjugation. In the other MOB families, plasmids containing NAP gene homologs were less than 10% (8%, MOBC; 9%, MOBF; 9%, MOBP; and 8%, MOBV). This phenomenon may also be related to the average size of the plasmids contained in each MOB family. MOBH (220 kb) and MOBQ (198 kb) were larger than MOBC (78 kb), MOBF (117 kb), MOBP (87 kb), and MOBV (149 kb). On the other hand, the average G+C content of all plasmids belonging to each MOB family was as follows: MOBC (52%), MOBF (52%), MOBH (51%), MOBP (53%), MOBQ (54%), and MOBV (46%). No relationship between the distribution of NAP gene homologs of each MOB family and the G+C content of plasmids was found.Table 7
MOBH-family plasmids of Gram-negative origina.
Plasmid nameAccession no.Source organismLength (nt)G+C content (%)bNAP gene homologIdentity (%)cQuery coverage (%)Subject startSubject end1NC_007949Polaromonassp. JS66636040557HU529961052607861NC_008573Shewanellasp. ANA-3278942462NC_007950Polaromonassp. JS66633800760ICEhin1056NC_011409Haemophilus influenzae5939339pAM04528NC_012693Salmonella enterica15821352HU57991406714333pAPEC-O1-RNC_009838Escherichia coli APEC O124138746pAR060302NC_012692Escherichia coli16653053HU57991575516021pAsa4NC_009349Aeromonas salmonicidasubsp.salmonicida A44916674953HU60992684426578pCAR1NC_004444Pseudomonas resinovorans19903556MvaT61987764077993NdpA36989539094395HU42999780998075pEC-IMPNC_012555Enterobacter cloacae31878248H-NS6499109370108969pEC-IMPQNC_012556Enterobacter cloacae32450348H-NS6499109370108969peH4HNC_012690Escherichia coli14810553HU57991406714333pHCM1NC_003384Salmonella entericasubsp.entericaserovar Typhi str. CT1821816048H-NS6199131861131460pIP1202NC_009141Yersinia pestisbv. Orientalis str. IP27518291353HU57991406714333pK29NC_010870Klebsiella pneumoniae26967446plasmid1NC_007901Rhodoferax ferrireducens T11825744754pMAK1NC_009981Salmonella entericasubsp.entericaserovar Choleraesuis20840947H-NS61996004659645pMAQU02NC_008739Marinobacter aquaeolei VT821329053pO111_1NC_013365Escherichia coli O111:H- str. 1112820460447H-NS61998017579774pP91278NC_008613Photobacterium damselaesubsp. Piscicida13152052HU5799125918126184pP99-018NC_008612Photobacterium damselaesubsp.piscicida15015751HU5799133314133580pRA1NC_012885Aeromonas hydrophila14396351HU58991557315839pRp12D01NC_012855Ralstonia pickettii 12D38977958HU3799321346321080pSN254NC_009140Salmonella entericasubsp.entericaserovar Newport str. SL25417647353HU57991406714333pTK9001NC_013930Thioalkalivibriosp. K90mix24025662pYR1NC_009139Yersinia ruckeri15803851HU57991507015336R27NC_002305Salmonella entericasubsp.entericaserovar Typhi18046146H-NS6199148225148626R478NC_005211Serratia marcescens27476246H-NS6499111747111346Rts1NC_003905Proteus vulgaris21718246aThis list is the result of a TBLASTN analysis using the 300 N-terminal amino acid sequence of protein TraI_R27 as a query under strict conditions (i.e., thresholds of 30% identity and 70% query coverage).bAverage G+C content of the plasmid.cReported TBLASTN identity to each NAP.Table 8
MOBQ-family plasmids of Gram-negative origina.
Plasmid nameAccession no.Source organismLength (nt)G+C content (%)bNAP gene homologIdentity (%)cQuery coverage (%)Subject startSubject end1NC_008242Chelativoranssp. BNC134393162HU41941339321336783NC_007617Nitrosospira multiformis ATCC 2519614159503NC_007961Nitrobacter hamburgensis X1412140862AtNC_003064Agrobacterium tumefaciensstr. C5854286857IHF3682112654112412CNC_010542Cyanothecesp. ATCC 511421468538ColE9-JNC_011977Escherichia coli757750DN1NC_002636Dichelobacter nodosus511262F plasmidNC_008036Sphingopyxis alaskensis RB22562854360p11745NC_013546Actinobacillus pleuropneumoniae548638p12494NC_010889Actinobacillus pleuropneumoniae1439333p1ABAYENC_010401Acinetobacter baumannii AYE564435p1META1NC_012807Methylobacterium extorquens AM14419568p1METDINC_012987Methylobacterium extorquens DM414150465p2007057NC_011897Salmonella entericasubsp.entericaserovar Bovismorbificans427047p2ABSDFNC_010396Acinetobacter baumannii SDF2501435p2ESCUMNC_011739Escherichia coli UMN0263380942p2META1NC_012809Methylobacterium extorquens AM13785865IHF44952836928635p3ABSDFNC_010398Acinetobacter baumannii SDF2492234p42aNC_007762Rhizobium etli CFN 4219422958p49879.1NC_006907Leptospirillum ferrooxidans2887858HU479932813015p49879.2NC_006909Leptospirillum ferrooxidans2801255HU48991585815592pAb5S9NC_009476Aeromonas bestiarum2471654pACRY07NC_009473Acidiphilium cryptum JF-5562958pAgK84NC_011994Agrobacterium radiobacter K844442054pAM5NC_008691Acidiphilium multivorum516158pAMI2NC_010847Paracoccus aminophilus1856362pAMI3NC_013513Paracoccus aminophilus557561pAPA01-030NC_013212Acetobacter pasteurianus IFO 3283-014996154pAPA01-040NC_013213Acetobacter pasteurianus IFO 3283-01320454pAtK84bNC_011990Agrobacterium radiobacter K8418466859IHF38865410953855pAtS4bNC_011991Agrobacterium vitis S413043556IHF47974488045152pAtS4cNC_011984Agrobacterium vitis S421162059HU4594141245140991pAtS4eNC_011981Agrobacterium vitis S463177557HU41944047640222Lrp3087460443460871Lrp3474425247424888pAV2NC_010310Acinetobacter venetianus1513536pBNC_010996Rhizobium etli CIAT 65242911158pBGR3NC_012847Bartonella grahamii as4aup2819236pBS512_5NC_010659Shigella boydii CDC 3083-94511446pCNC_010997Rhizobium etli CIAT 652109152361Lrp4688617696618130Lrp4290609059608619Lrp3995417738418202Lrp4279714804715193Lrp3993406570407025pCAUL01NC_010335Caulobactersp. K3123364967HU44999759897329Lrp3489182479182042pCAUL02NC_010333Caulobactersp. K3117787864pCCK1900NC_011378Pasteurella multocida1022661pCCK381NC_006994Pasteurella multocida1087461pCFPG4NC_011563Candidatus Azobacteroides pseudotrichonymphagenomovar. CFP2414944pCHE-ANC_012006Enterobacter cloacae756060pColE8NC_012882Escherichia coli675151pCROD3NC_013719Citrobacter rodentium ICC168391051pCT02021853_74NC_011204Salmonella entericasubsp.entericaserovar Dublin str. CT_020218537455149pCVM19633_4NC_011093Salmonella entericasubsp.entericaserovar Schwarzengrund str. CVM19633458548pDSHI01NC_009955Dinoroseobacter shibae DFL 1219050660pET09NC_010695Erwinia tasmaniensis Et1/99929947pGDIA01NC_011367Gluconacetobacter diazotrophicus PAl 52745559pGOX3NC_006674Gluconobacter oxydans 621H1454756pHCG3NC_005873Oligotropha carboxidovorans OM513305861pHRM2aNC_012109Desulfobacterium autotrophicum HRM26870942pIGJC156NC_009781Escherichia coli514647pIGMS5NC_010883Escherichia coli675051pIGWZ12NC_010885Escherichia coli407250pISP3NC_013970Sphingomonassp. MM-14339863pJD4NC_002098Neisseria gonorrhoeae742638plasmid1NC_007801Jannaschiasp. CCS18607258pLD-TEX-KLNC_009966Fluoribacter dumoffii6651239pMACNC_006877Acinetobacter baumannii954035pMAS2027NC_013503Escherichia coli4264443pMbo4.6NC_013500Moraxella bovis465839pMCHL01NC_011758Methylobacterium chloromethanicum CM438020766pMG160NC_004527Rhodobacter blasticus343167pMG828-2NC_008487Escherichia coli409150pMG828-4NC_008489Escherichia coli746248pMMCU1NC_013056Acinetobacter calcoaceticus877135pMMCU2NC_013506Acinetobacter baumannii1027036pMRAD01NC_010510Methylobacterium radiotolerans JCM 283158616470pMS260NC_005312Actinobacillus pleuropneumoniae812461pNGR234aNC_000914Rhizobiumsp. NGR23453616559Lrp4170197189196845Lrp3089188867188430pNGR234bNC_012586Rhizobiumsp. NGR234243003362Lrp4690656547656107Lrp4585667494667913Lrp439010380201038463Lrp4485682796683215Lrp389624008492401319Lrp4479709104708715Lrp41892833628761Lrp338911089001109337Lrp3690703213702764Lrp327711129531112582pNL2NC_009427Novosphingobium aromaticivorans DSM 1244448726866pO111_4NC_013367Escherichia coli O111:H- str. 11128814050pO26-S4NC_011228Escherichia coli675851pOLA52NC_010378Escherichia coli5160246pOU1114NC_010421Salmonella entericasubsp.entericaserovar Dublin3459542pOU1115NC_010422Salmonella entericasubsp.entericaserovar Dublin7458949pPNC_003455Salmonella entericasubsp.entericaserovarEnteritidis430150pP742405NC_011733Cyanothecesp. PCC 74241808338pP742406NC_011734Cyanothecesp. PCC 74241521940pPMA4326CNC_005921Pseudomonas syringaepv.maculicola824453pPNAP07NC_008763Polaromonas naphthalenivorans CJ2989857pPRO2NC_008608Pelobacter propionicus DSM 23793072256pPT1NC_002143Comamonas testosteroni1539856pR132501NC_012848Rhizobium leguminosarumbv. trifolii WSM132582892460Lrp4788234905234471Lrp4486386338386760Lrp3993645542645087Lrp4279147165146776pR132502NC_012858Rhizobium leguminosarumbv. trifolii WSM132566097361pR132503NC_012853Rhizobium leguminosarumbv. trifolii WSM132551608859HU4794300662300916pR132504NC_012852Rhizobium leguminosarumbv. trifolii WSM132535031261pR132505NC_012854Rhizobium leguminosarumbv. trifolii WSM132529478260pRFNC_007110Rickettsia felis URRWXCal26282934pRFdeltaNC_007111Rickettsia felis URRWXCal23926333pRi1724NC_002575Agrobacterium rhizogenes21759457pRi2659NC_010841Agrobacterium rhizogenes18546258pRL10NC_008381Rhizobium leguminosarumbv. viciae 384148813560pRL11NC_008384Rhizobium leguminosarumbv. viciae 384168420261pRL12NC_008378Rhizobium leguminosarumbv. viciae 384187002161Lrp4688599116598682Lrp4388658287658718Lrp39934560145146Lrp4279450080449691pRL7NC_008382Rhizobium leguminosarumbv. viciae 384115156458HU48942048420230pRL8NC_008383Rhizobium leguminosarumbv. viciae 384114746359Lrp33877076370344pRLG201NC_011368Rhizobium leguminosarumbv. trifolii WSM2304126610560Lrp4589917573917136Lrp44854199842417Lrp4479473039472650Lrp409311621461161691Lrp409311509391150484Lrp3288707587707162pRMNC_010927Rickettsia monacensis2348632pSC101NC_002056Salmonella entericasubsp.entericaserovar Typhimurium926351pSE11-6NC_011411Escherichia coli SE11408249pSE34NC_010860Salmonella entericasubsp.entericaserovar Enteritidis3295041pSMED01NC_009620Sinorhizobium medicae WSM419157095162Lrp4077143180143557Lrp3489574284573847pSMED02NC_009621Sinorhizobium medicae WSM419124540860Lrp4291556486556932Lrp4091842324842758Lrp31872234521917pSmeSM11aNC_013545Sinorhizobium meliloti14417060Lrp46967044970922pSmeSM11bNC_010865Sinorhizobium meliloti SM1118125159pSMS35_4NC_010486Escherichia coli SMS-3-5407450pSx-QyyNC_006826Sphingobium xenophagum568356pSymANC_003037Sinorhizobium meliloti 1021135422660LrppSymBNC_003078Sinorhizobium meliloti 1021168333362Lrp3890440778440335Lrp36892955529992pTB3NC_008388Roseobacter denitrificans OCh 1141657555pTcM1NC_010600Acidithiobacillus caldus6515857IHF56892518625449pTiS4NC_011982Agrobacterium vitis S425882457HU41942735627102HU40948340883154Lrp42799692097309pTi-SAKURANC_002147Agrobacterium tumefaciens20647956HU44949576395509pUT1NC_014005Sphingobium japonicum UT26S3177664pUT2NC_014009Sphingobium japonicum UT26S539861pXAUT01NC_009717Xanthobacter autotrophicus Py231616465pXCV19NC_007505Xanthomonas campestrispv.vesicatoriastr. 85-101914660pXF51NC_002490Xylella fastidiosa 9a5c5115850pYAN-1NC_008246Sphingobium yanoikuyae518262pYAN-2NC_008247Sphingobium yanoikuyae492464RSF1010NC_001740Escherichia coli868461Symbiotic plasmid p42dNC_004041Rhizobium etli CFN 4237125458TiNC_002377Agrobacterium tumefaciens19414055IHF4397180164180436TiNC_003065Agrobacterium tumefaciensstr. C5821423357HU4494139735139481Ti plasmid pTiBo542NC_010929Agrobacterium tumefaciens24497855IHF3686209743209489IHF4598187204187479UnnamedNC_011143Phenylobacterium zucineum HLK138297669aThis list is the result of a TBLASTN analysis using the 300 N-terminal amino acid sequence of protein MobA_RSF1010 as a query under strict conditions (i.e., thresholds of 30% identity and 70% query coverage).bAverage G+C content of the plasmid.cReported TBLASTN identity to each NAP.


## 3.6. Conclusions

We compared the distribution of NAP gene homologs among plasmids and plasmid features. Larger plasmids frequently had NAP gene homologs, possibly to maintain themselves and host cell fitness. Plasmids with NAP gene homologs also frequently carried relaxase genes. Although this may be related to their relatively larger sizes, together with the fact that NAPs affect global gene regulation, it is likely that NAPs contribute to plasmid transmission. Considering the fact that NAPs encoded on plasmids actually help the host cell to integrate newly acquired genes into host regulatory networks [14, 15], large plasmids with NAP gene homologs may be generally more beneficial not only for the host cell, but also for their own existence.NAP homologs encoded on plasmids can interact with different types of NAPs encoded on the host chromosome and cooperatively regulate host transcriptional networks. Understanding these mechanisms in more detail will shed light on the meanings of the distributions of NAPs on plasmids and chromosomes. Comprehensive analysis of their binding sites in the host and plasmid genomes will help us to understand the relationships between G+C content and the presence of NAPs. Such information will explain how bacteria adapt and evolve by acquiring foreign genes by HGT.



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*Source: 685015-2011-01-24.xml*