Distribution of genes encoding nucleoid-associated protein homologs in plasmids.

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.

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.

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 (Table 1). 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 name	Accession no.	Source organism	Length (nt)	G+C content (%)b	Identity (%)c	Query coverage (%)	Subject start	Subject end	Classificationd	MOB familye
1	NC_013972	Erwinia amylovora ATCC 49946	28243	50	66	99	3129	2728	−
pAsa5	NC_009350	Aeromonas salmonicida subsp. salmonicida A449	155098	54	46	99	941	534	−	MOBF
					47	99	16890	16483
pAsal5	NC_009352	Aeromonas salmonicida subsp. salmonicida	18536	54	46	99	12285	12692	−
pEA29	NC_013957	Erwinia amylovora CFBP1430	28259	50	66	99	3129	2728	−
pEA29	NC_005706	Erwinia amylovora	28185	50	64	99	2991	2590	−
pEC-IMP	NC_012555	Enterobacter cloacae	318782	48	64	99	109370	108969	−	MOBH
pEC-IMPQ	NC_012556	Enterobacter cloacae	324503	48	64	99	109370	108969	−	MOBH
pEJ30	NC_004834	Erwinia sp. Ejp 556	29593	50	66	99	4651	4250	−
pEP36	NC_013263	Erwinia pyrifoliae Ep1/96	35909	50	66	99	25040	25441	−
pEP36	NC_004445	Erwinia pyrifoliae Ep1/96	35904	50	64	98	4675	4280	−
pET45	NC_010699	Erwinia tasmaniensis Et1/99	44694	51	52	93	37435	37809	−	MOBF
pET49	NC_010697	Erwinia tasmaniensis Et1/99	48751	44	36	94	30821	31204	−
pHCM1	NC_003384	Salmonella enterica subsp. enterica serovar Typhi str. CT18	218160	48	61	99	131861	131460	−	MOBH
pK2044	NC_006625	Klebsiella pneumoniae NTUH-K2044	224152	50	67	99	35717	36112	−
plasmid_153kb	NC_009705	Yersinia pseudotuberculosis IP 31758	153140	40	44	100	139846	140265	−
pLVPK	NC_005249	Klebsiella pneumoniae	219385	50	67	99	114397	114792	−
pMAK1	NC_009981	Salmonella enterica subsp. enterica serovar Choleraesuis	208409	47	61	99	60046	59645	−	MOBH
pO111_1	NC_013365	Escherichia coli O111:H- str. 11128	204604	47	61	99	80175	79774	−	MOBH
pSG1	NC_007713	Sodalis glossinidius str. “morsitans”	83306	49	43	97	2533	2922	−
R27	NC_002305	Salmonella enterica subsp. enterica serovar Typhi	180461	46	61	99	148225	148626	−	MOBH
R478	NC_005211	Serratia marcescens	274762	46	64	99	111747	111346	−	MOBH
Unnamed	NC_011148	Salmonella enterica subsp. enterica serovar Agona str. SL483	37978	41	43	95	7671	7288	−

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 flexneri 2a 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 name	Accession no.	Source organism	Length (nt)	G+C content (%)b	Identity (%)c	Query coverage (%)	Subject start	Subject end	Classificationd	MOB familye
1	NC_006823	Aromatoleum aromaticum EbN1	207355	58	55	99	186175	185909	−
1	NC_007949	Polaromonas sp. JS666	360405	57	52	99	61052	60786	−	MOBH
1	NC_008010	Deinococcus geothermalis DSM 11300	574127	66	38	97	550805	550545	+
1	NC_008503	Lactococcus lactis subsp. cremoris SK11	14041	34	37	94	9732	10007	+	MOBP
1	NC_008242	Chelativorans sp. BNC1	343931	62	41	94	133932	133678	−	MOBQ
2	NC_012529	Deinococcus deserti VCD115	314317	64	38	93	269648	269899	+
3	NC_012528	Deinococcus deserti VCD115	396459	61	40	96	8700	8957	+
Megaplasmid	NC_007974	Cupriavidus metallidurans CH34	2580084	64	51	99	1393415	1393149	−	MOBV
Megaplasmid	NC_005863	Desulfovibrio vulgaris str. Hildenborough	202301	66	31	98	5502	5765	−
Megaplasmid pDF308	NC_013940	Deferribacter desulfuricans SSM1	308544	24	41	100	253817	253548	−
Megaplasmid pHG1	NC_005241	Ralstonia eutropha H16	452156	62	48	99	343060	342791	−
p49879.1	NC_006907	Leptospirillum ferrooxidans	28878	58	47	99	3281	3015	−	MOBQ
p49879.2	NC_006909	Leptospirillum ferrooxidans	28012	55	48	99	15858	15592	−	MOBQ
pAH187_270	NC_011655	Bacillus cereus AH187	270082	34	59	100	113139	112870	+
pAH820_272	NC_011777	Bacillus cereus AH820	272145	34	58	100	153060	152791	+
pAM04528	NC_012693	Salmonella enterica	158213	52	57	99	14067	14333	−	MOBH
pAOVO01	NC_008765	Acidovorax sp. JS42	72689	62	46	100	65140	64871	−	MOBF
pAPA01-011	NC_013210	Acetobacter pasteurianus IFO 3283-01	191799	53	47	100	154736	154467	−
					46	99	38442	38708
pAR060302	NC_012692	Escherichia coli	166530	53	57	99	15755	16021	−	MOBH
pAsa4	NC_009349	Aeromonas salmonicida subsp. salmonicida A449	166749	53	60	99	26844	26578	−	MOBH
pAtS4c	NC_011984	Agrobacterium vitis S4	211620	59	45	94	141245	140991	−	MOBQ
pAtS4e	NC_011981	Agrobacterium vitis S4	631775	57	41	94	40476	40222	−	MOBQ
pBc239	NC_011973	Bacillus cereus Q1	239246	33	52	100	191895	192164	+
pBF9343	NC_006873	Bacteroides fragilis NCTC 9343	36560	32	35	92	15803	15558	−	MOBP
pBPHY01	NC_010625	Burkholderia phymatum STM815	1904893	62	43	99	826527	826252	−
pBPHY02	NC_010627	Burkholderia phymatum STM815	595108	59	45	99	98625	98359	−
pBtoxis	NC_010076	Bacillus thuringiensis serovar israelensis	127923	32	52	99	77382	77648	+
pBWB401	NC_010180	Bacillus weihenstephanensis KBAB4	417054	34	59	100	338347	338078	+
pCAR1	NC_004444	Pseudomonas resinovorans	199035	56	42	99	97809	98075	−	MOBH
pCAUL01	NC_010335	Caulobacter sp. K31	233649	67	44	99	97598	97329	−	MOBQ
pCER270	NC_010924	Bacillus cereus	270082	34	59	100	169548	169279	+
pDBORO	NC_009137	Lactococcus lactis subsp. lactis bv. diacetylactis	16404	35	37	94	16387	16112	+
pDVUL01	NC_008741	Desulfovibrio vulgaris DP4	198504	66	31	98	198317	198054	−
peH4H	NC_012690	Escherichia coli	148105	53	57	99	14067	14333	−	MOBH
pG9842_209	NC_011775	Bacillus cereus G9842	209488	30	60	100	88828	88559	+
pH308197_258	NC_011339	Bacillus cereus H3081.97	258484	34	59	100	83033	83302	+
pHD5AT	NC_012752	Candidatus Hamiltonella defensa 5AT (Acyrthosiphon pisum)	59032	45	45	99	14981	15247	−	MOBP
pIP1202	NC_009141	Yersinia pestis bv. Orientalis str. IP275	182913	53	57	99	14067	14333	−	MOBH
plasmid 2	NC_007972	Cupriavidus metallidurans CH34	171459	61	46	99	125530	125261	−
pMOL28	NC_006525	Cupriavidus metallidurans CH34	171461	61	46	99	51529	51798	−
pMP118	NC_007930	Lactobacillus salivarius UCC118	242436	32	54	99	56763	56497	+	MOBV
pNPUN02	NC_010632	Nostoc punctiforme PCC 73102	254918	41	44	99	74804	74538	−	MOBV
pOANT02	NC_009670	Ochrobactrum anthropi ATCC 49188	101491	59	49	94	32700	32446	−
pP91278	NC_008613	Photobacterium damselae subsp. piscicida	131520	52	57	99	125918	126184	−	MOBH
pP99-018	NC_008612	Photobacterium damselae subsp. piscicida	150157	51	57	99	133314	133580	−	MOBH
pPER272	NC_010921	Bacillus cereus	272145	34	58	100	153060	152791	+
pPMA4326A	NC_005918	Pseudomonas syringae pv. maculicola	46697	55	42	99	1520	1786	−
pPMA4326B	NC_005919	Pseudomonas syringae pv. maculicola	40110	55	45	99	1457	1723	−
pQBR103	NC_009444	Pseudomonas fluorescens SBW25	425094	53	51	99	182862	183128	−
pR132503	NC_012853	Rhizobium leguminosarum bv. trifolii WSM1325	516088	59	47	94	300662	300916	−	MOBQ
pRA1	NC_012885	Aeromonas hydrophila	143963	51	58	99	15573	15839	−	MOBH
pRALTA	NC_010529	Cupriavidus taiwanensis	557200	60	46	98	153542	153276	−
pREB1	NC_009926	Acaryochloris marina MBIC11017	374161	47	46	100	339743	340012	−	MOBF
pREB2	NC_009927	Acaryochloris marina MBIC11017	356087	45	48	100	57583	57852	−	MOBF
pREB3	NC_009928	Acaryochloris marina MBIC11017	273121	45	46	100	234682	234951	−	MOBF
					42	100	243339	243608
pRL7	NC_008382	Rhizobium leguminosarum bv. viciae 3841	151564	58	48	94	20484	20230	−	MOBQ
pRLG203	NC_011370	Rhizobium leguminosarum bv. trifolii WSM2304	308747	58	49	94	141121	140867	−
pRp12D01	NC_012855	Ralstonia pickettii 12D	389779	58	37	99	321346	321080	−	MOBH
pSG2	NC_007184	Sodalis glossinidius	27240	45	45	86	10072	9845	−
pSG3	NC_007186	Sodalis glossinidius	19201	51	51	100	13812	13543	−
pSN254	NC_009140	Salmonella enterica subsp. enterica serovar Newport str. SL254	176473	53	57	99	14067	14333	−	MOBH
pTiS4	NC_011982	Agrobacterium vitis S4	258824	57	41	94	27356	27102	−	MOBQ
					40	94	83408	83154
pTi-SAKURA	NC_002147	Agrobacterium tumefaciens	206479	56	44	94	95763	95509	−	MOBQ
pVSAL840	NC_011311	Aliivibrio salmonicida LFI1238	83540	40	60	99	31361	31627	−	MOBF
					58	99	77350	77084
pYR1	NC_009139	Yersinia ruckeri	158038	51	57	99	15070	15336	−	MOBH
Ti	NC_003065	Agrobacterium tumefaciens str. C58	214233	57	44	94	139735	139481	−	MOBQ

aThis 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 name	Accession no.	Source organism	Length (nt)	G+C content (%)b	Identity (%)c	Query coverage (%)	Subject start	Subject end	Classificationd	MOB familye
At	NC_003064	Agrobacterium tumefaciens str. C58	542868	57	36	82	112654	112412	−	MOBQ
Megaplasmid	NC_012811	Methylobacterium extorquens AM1	1261460	68	33	94	720582	720860	−
p2META1	NC_012809	Methylobacterium extorquens AM1	37858	65	44	95	28369	28635	−	MOBQ
pAACI01	NC_013206	Alicyclobacillus acidocaldarius subsp. acidocaldarius DSM 446	91726	54	43	80	62668	62432	+
pACHL01	NC_011879	Arthrobacter chlorophenolicus A6	426858	64	32	92	408818	408546	+
pALVIN02	NC_013862	Allochromatium vinosum DSM 180	39929	53	60	98	10902	10627	−
pAph01	NC_013193	Candidatus Accumulibacter phosphatis clade IIA str. UW-1	167595	62	56	95	144197	144463	−	MOBP
pAph03	NC_013191	Candidatus Accumulibacter phosphatis clade IIA str. UW-1	37695	59	58	97	5412	5140	−
pAtK84b	NC_011990	Agrobacterium radiobacter K84	184668	59	38	86	54109	53855	−	MOBQ
pAtK84c	NC_011987	Agrobacterium radiobacter K84	388169	57	43	93	340807	340532	−
					46	93	10327	10052
pAtS4b	NC_011991	Agrobacterium vitis S4	130435	56	47	97	44880	45152	−	MOBQ
pBBta01	NC_009475	Bradyrhizobium sp. BTAi1	228826	61	39	86	6642	6388	−
pBFY46	NC_006297	Bacteroides fragilis YCH46	33716	34	35	89	25098	25343	−	MOBP
pBIND01	NC_010580	Beijerinckia indica subsp. indica ATCC 9039	181736	56	36	77	179816	179601	−	MOBF
pCHQ1	NC_014007	Sphingobium japonicum UT26S	190974	63	36	90	63111	63377	−
pGLOV01	NC_010815	Geobacter lovleyi SZ	77113	53	38	92	41196	41468	−
pM44601	NC_010373	Methylobacterium sp. 4-46	57951	65	35	97	7806	7534	−
pMPOP01	NC_010727	Methylobacterium populi BJ001	25164	65	49	93	10635	10375	−
pMRAD03	NC_010514	Methylobacterium radiotolerans JCM 2831	42985	63	38	94	26778	26515	−	MOBF
pMRAD04	NC_010517	Methylobacterium radiotolerans JCM 2831	37743	64	38	94	10763	10500	−
pPRO1	NC_008607	Pelobacter propionicus DSM 2379	202397	48	41	94	129679	129957	−
pRSPA01	NC_009429	Rhodobacter sphaeroides ATCC 17025	877879	68	49	97	783519	783791	−
pSWIT01	NC_009507	Sphingomonas wittichii RW1	310228	64	40	95	106554	106820	−	MOBF
					36	92	35341	35069
pTcM1	NC_010600	Acidithiobacillus caldus	65158	57	56	89	25186	25449	−	MOBP, MOBQ
pXCV183	NC_007507	Xanthomonas campestris pv. vesicatoria str. 85-10	182572	60	33	95	138753	138490	−
Ti	NC_002377	Agrobacterium tumefaciens	194140	55	43	97	180164	180436	−	MOBQ
Ti plasmid pTiBo542	NC_010929	Agrobacterium tumefaciens	244978	55	36	86	209743	209489	−	MOBQ
					45	98	187204	187479

aThis 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 name	Accession no.	Source organism	Length (nt)	G+C content (%)b	Identity (%)c	Query coverage (%)	Subject start	Subject end	Classificationd	MOB familye
1	NC_008688	Paracoccus denitrificans PD1222	653815	67	41	92	252075	251623	−
					42	93	464218	464673
					36	96	639341	639811
					37	85	110140	109724
A	NC_009007	Rhodobacter sphaeroides 2.4.1	114045	69	39	93	30241	29789	−	MOBF
B	NC_007488	Rhodobacter sphaeroides 2.4.1	114178	70	43	96	81861	81385	−
bglu_1p	NC_012723	Burkholderia glumae BGR1	133591	61	36	90	124017	123577	−
Megaplasmid	NC_008043	Ruegeria sp. TM1040	821788	59	41	84	143820	144233	−
					41	91	687257	687706
					36	91	734136	733690
Megaplasmid	NC_007974	Cupriavidus metallidurans CH34	2580084	64	44	88	1171245	1170814	−	MOBV
					40	91	1169702	1169256
					38	97	1586726	1586250
Megaplasmid	NC_006569	Ruegeria pomeroyi DSS-3	491611	63	36	88	356303	355869	−	MOBC
Megaplasmid	NC_007336	Ralstonia eutropha JMP134	634917	61	35	93	377503	377045	−
p42e	NC_007765	Rhizobium etli CFN 42	505334	62	34	71	255037	255384	−
p42f	NC_007766	Rhizobium etli CFN 42	642517	61	45	88	436907	437341	−
					43	91	406350	405901
					41	85	491383	491799
					39	95	210634	211098
					39	96	199426	199899
pAB510a	NC_013855	Azospirillum sp. B510	1455109	68	57	88	274908	275342	−
					44	95	979549	980013
					32	94	1180335	1179874
pAB510b	NC_013856	Azospirillum sp. B510	723779	67	44	84	471830	472243	−
					32	94	318139	318600
pAB510c	NC_013857	Azospirillum sp. B510	681723	67	45	85	408064	407645	−
					34	91	36385	36834
pAB510d	NC_013858	Azospirillum sp. B510	628837	68	44	79	472768	472379	−
					40	90	323184	322741
					37	87	281438	281866
					30	85	619027	618623
pAtS4e	NC_011981	Agrobacterium vitis S4	631775	57	30	87	460443	460871	−	MOBQ
					34	74	425247	424888
pBPHY01	NC_010625	Burkholderia phymatum STM815	1904893	62	46	85	1153608	1154027	−
pBPHY02	NC_010627	Burkholderia phymatum STM815	595108	59	41	91	271795	271346	−
pC	NC_010997	Rhizobium etli CIAT 652	1091523	61	46	88	617696	618130	−	MOBQ
					42	90	609059	608619
					39	95	417738	418202
					42	79	714804	715193
					39	93	406570	407025
pCAUL01	NC_010335	Caulobacter sp. K31	233649	67	34	89	182479	182042	−	MOBQ
pEST4011	NC_005793	Achromobacter denitrificans	76958	62	58	88	41224	40793	−	MOBP
					58	88	34233	33802
pGMI1000MP	NC_003296	Ralstonia solanacearum GMI1000	2094509	67	43	98	1737958	1738437	−
					46	93	822030	821572
pHV4	NC_013966	Haloferax volcanii DS2	635786	62	33	71	401763	401410	Archaea
pIJB1	NC_013666	Burkholderia cepacia	99448	63	58	88	74907	75338	−	MOBP
pK2044	NC_006625	Klebsiella pneumoniae NTUH-K2044	224152	50	33	90	194643	195086	−
pLVPK	NC_005249	Klebsiella pneumoniae	219385	50	33	90	46236	46679	−
pMLa	NC_002679	Mesorhizobium loti MAFF303099	351911	59	32	93	185603	185148	−
					30	89	207314	206877
pMLb	NC_002682	Mesorhizobium loti MAFF303099	208315	60	37	93	24632	24177	−
pNGR234a	NC_000914	Rhizobium sp. NGR234	536165	58	41	70	197189	196845	−	MOBQ
					30	89	188867	188430
pNGR234b	NC_012586	Rhizobium sp. NGR234	2430033	62	46	90	656547	656107	−	MOBQ
					45	85	667494	667913
					43	90	1038020	1038463
					44	85	682796	683215
					38	96	2400849	2401319
					44	79	709104	708715
					41	89	28336	28761
					33	89	1108900	1109337
					36	90	703213	702764
					32	77	1112953	1112582
pPNAP04	NC_008760	Polaromonas naphthalenivorans CJ2	143747	59	35	90	142511	142068	−
pR132501	NC_012848	Rhizobium leguminosarum bv. trifolii WSM1325	828924	60	47	88	234905	234471	−	MOBQ
					44	86	386338	386760
					39	93	645542	645087
					42	79	147165	146776
pRALTA	NC_010529	Cupriavidus taiwanensis	557200	60	38	91	465839	465393	−
pRHL1	NC_008269	Rhodococcus jostii RHA1	1123075	65	36	91	854207	854656	+
					33	84	783666	783253
pRL12	NC_008378	Rhizobium leguminosarum bv. viciae 3841	870021	61	46	88	599116	598682	−	MOBQ
					43	88	658287	658718
					39	93	45601	45146
					42	79	450080	449691
pRL8	NC_008383	Rhizobium leguminosarum bv. viciae 3841	147463	59	33	87	70763	70344	−	MOBQ
pRLG201	NC_011368	Rhizobium leguminosarum bv. trifolii WSM2304	1266105	60	45	89	917573	917136	−	MOBQ
					44	85	41998	42417
					44	79	473039	472650
					40	93	1162146	1161691
					40	93	1150939	1150484
					32	88	707587	707162
pRSKD131A	NC_011962	Rhodobacter sphaeroides KD131	157345	70	42	96	148295	147819	−
pRSKD131B	NC_011960	Rhodobacter sphaeroides KD131	103355	70	39	93	98400	97948	−
pRSPA01	NC_009429	Rhodobacter sphaeroides ATCC 17025	877879	68	40	90	31309	30866	−
					39	88	659383	658952
pRSPH01	NC_009040	Rhodobacter sphaeroides ATCC 17029	122606	70	39	93	118088	118540	−
pSMED01	NC_009620	Sinorhizobium medicae WSM419	1570951	61	40	77	143180	143557	−	MOBQ
					34	89	574284	573847
pSMED02	NC_009621	Sinorhizobium medicae WSM419	1245408	60	42	91	556486	556932	−	MOBQ
					40	91	842324	842758
					31	87	22345	21917
pSMED03	NC_009622	Sinorhizobium medicae WSM419	219313	60	46	95	105044	105508	−
pSmeSM11a	NC_013545	Sinorhizobium meliloti	144170	60	46	96	70449	70922	−	MOBQ
pSymA	NC_003037	Sinorhizobium meliloti 1021	1354226	60	43	89	1060699	1060262	−	MOBQ
pSymB	NC_003078	Sinorhizobium meliloti 1021	1683333	62	38	90	440778	440335	−	MOBQ
					36	89	29555	29992
pTiS4	NC_011982	Agrobacterium vitis S4	258824	57	42	79	96920	97309	−	MOBQ
Unnamed	NC_011961	Thermomicrobium roseum DSM 5159	917738	66	30	85	736739	737146	−	MOBP

aThis 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 name	Accession no.	Source organism	Length (nt)	G+C content (%)b	Identity (%)c	Query coverage (%)	Subject start	Subject end	Classificationd	MOB familye
MvaT
pCAR1	NC_004444	Pseudomonas resinovorans	199035	56	61	98	77640	77993	−	MOBH
pQBR103	NC_009444	Pseudomonas fluorescens SBW25	425094	53	61	96	98076	97717	−
pWW53	NC_008275	Pseudomonas putida	107929	57	61	98	8415	8768	−
NdpA
p0908	NC_010113	Vibrio sp. 0908	81413	49	51	99	79731	78736	−
pCAR1	NC_004444	Pseudomonas resinovorans	199035	56	36	98	95390	94395	−	MOBH
pQBR103	NC_009444	Pseudomonas fluorescens SBW25	425094	53	31	99	161413	160400	−

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 Figure 1(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.

Figure 1

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.

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) (Figure 1(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 name	Accession no.	Source organism	Length (nt)	G+C content (%)b	NAP gene homolog	Identity (%)c	Query coverage (%)	Subject start	Subject end	MOB familyd
55989p	NC_011752	Escherichia coli 55989	72482	46		53	92	10025	9828
NR1	NC_009133	Escherichia coli	94289	52		53	92	87193	87390	MOBF
p1658/97	NC_004998	Escherichia coli	125491	51		55	92	36419	36616	MOBF
p1ESCUM	NC_011749	Escherichia coli UMN026	122301	50		53	92	53508	53311	MOBF
p2ESCUM	NC_011739	Escherichia coli UMN026	33809	42		62	90	7682	7488	MOBQ
p53638_226	NC_010719	Escherichia coli 53638	225683	48		55	92	67615	67418	MOBF
pAPEC-O1-R	NC_009838	Escherichia coli APEC O1	241387	46		50	92	61389	61586	MOBH
pAPEC-O2-ColV	NC_007675	Escherichia coli	184501	49		55	92	3882	3685	MOBF
pAPEC-O2-R	NC_006671	Escherichia coli	101375	53		53	92	4856	4659	MOBF
pBS512_211	NC_010660	Shigella boydii CDC 3083-94	210919	46		55	89	190719	190910	MOBF
pBS512_33	NC_010657	Shigella boydii CDC 3083-94	33103	41		62	90	2894	3088
pC15-1a	NC_005327	Escherichia coli	92353	53		53	92	87490	87687	MOBF
pCP301	NC_004851	Shigella flexneri 2a str. 301	221618	46		55	92	207828	208025	MOBF
pCROD1	NC_013717	Citrobacter rodentium ICC168	54449	47		56	92	53220	53417
pCROD2	NC_013718	Citrobacter rodentium ICC168	39265	42		62	90	15526	15332
pCT02021853_74	NC_011204	Salmonella enterica subsp. enterica serovar Dublin str. CT_02021853	74551	49		62	90	48482	48288	MOBQ
pCTX-M3	NC_004464	Citrobacter freundii	89468	51		38	71	26136	26294	MOBP
			89468			31	96	40648	40439
pCTXM360	NC_011641	Klebsiella pneumoniae	68018	51		38	71	64551	64709	MOBP
			68018			31	96	10927	10718
pCVM29188_146	NC_011076	Salmonella enterica subsp. enterica serovar Kentucky str. CVM29188	146811	49		53	92	18755	18558	MOBF
pEC14_114	NC_013175	Escherichia coli	114222	51		53	92	113985	114182	MOBF
pEC-IMP	NC_012555	Enterobacter cloacae	318782	48	H-NS	50	92	60491	60688	MOBH
pEC-IMPQ	NC_012556	Enterobacter cloacae	324503	48	H-NS	50	92	60491	60688	MOBH
pEG356	NC_013727	Shigella sonnei	70275	52		53	92	69444	69641	MOBF
pEK499	NC_013122	Escherichia coli	117536	53		53	92	41985	42182
pEK516	NC_013121	Escherichia coli	64471	53		53	92	31410	31213
pEL60	NC_005246	Erwinia amylovora	60145	51		38	71	23187	23345	MOBP
			60145			31	96	37863	37654
pEntH10407	NC_013507	Escherichia coli ETEC H10407	67094	51		55	78	43421	43254	MOBF
pHCM1	NC_003384	Salmonella enterica subsp. enterica serovar Typhi str. CT18	218160	48	H-NS	47	100	105911	106117	MOBH
pK2044	NC_006625	Klebsiella pneumoniae NTUH-K2044	224152	50	H-NS, Lrp	45	85	143331	143528
pK29	NC_010870	Klebsiella pneumoniae	269674	46		50	92	59322	59519	MOBH
pKF3-70	NC_013542	Klebsiella pneumoniae	70057	52		53	92	15967	15770	MOBF
pKF3-94	NC_013950	Klebsiella pneumoniae	94219	52		58	96	9596	9390	MOBF
pKP187	NC_011282	Klebsiella pneumoniae 342	187922	47		64	96	110083	109877
			187922			42	89	1550	1344
pKPN3	NC_009649	Klebsiella pneumoniae subsp. pneumoniae MGH 78578	175879	52		59	97	56930	56721	MOBF
plasmid_153 kb	NC_009705	Yersinia pseudotuberculosis IP 31758	153140	40	H-NS	69	93	63342	63542
			153140			56	92	49734	49931
pLVPK	NC_005249	Klebsiella pneumoniae	219385	50	H-NS, Lrp	61	97	148056	147847
			219385			45	85	214828	215025
pMAK1	NC_009981	Salmonella enterica subsp. enterica serovar Choleraesuis	208409	47	H-NS	47	100	49208	49414	MOBH
pMAS2027	NC_013503	Escherichia coli	42644	43		62	90	19685	19491	MOBQ
pO103	NC_013354	Escherichia coli O103:H2 str. 12009	75546	49		55	92	51727	51924	MOBF
pO111_1	NC_013365	Escherichia coli O111:H- str. 11128	204604	47	H-NS	47	100	66925	67131	MOBH
pO111_3	NC_013366	Escherichia coli O111:H- str. 11128	77690	50		55	92	11975	12172	MOBF
pO157	NC_013010	Escherichia coli O157:H7 str. TW14359	94601	48		55	92	70792	70989
pO157	NC_011350	Escherichia coli O157:H7 str. EC4115	94644	48		55	92	54735	54932
pO157	NC_007414	Escherichia coli O157:H7 EDL933	92077	48		55	92	1667	1864
pO157	NC_002128	Escherichia coli O157:H7 str. Sakai	92721	48		55	92	71183	71380
pO26I	NC_011812	Escherichia coli	72946	51		53	92	66608	66805	MOBF
pO86A1	NC_008460	Escherichia coli	120730	49		55	92	101598	101795	MOBF
pOLA52	NC_010378	Escherichia coli	51602	46		62	90	12114	11920	MOBQ
pOU1114	NC_010421	Salmonella enterica subsp. enterica serovar Dublin	34595	41		62	90	5446	5252	MOBQ
pOU1115	NC_010422	Salmonella enterica subsp. enterica serovar Dublin	74589	49		62	90	37246	37052	MOBQ
pSB4_227	NC_007608	Shigella boydii Sb227	126697	47		55	92	110688	110885	MOBF
pSE11-1	NC_011419	Escherichia coli SE11	100021	50		56	92	58407	58210	MOBP
pSE34	NC_010860	Salmonella enterica subsp. enterica serovar Enteritidis	32950	41		62	90	21875	22069	MOBQ
pSFO157	NC_009602	Escherichia coli	121239	50		52	75	1709	1870	MOBF
pSG1	NC_007713	Sodalis glossinidius str. “morsitans”	83306	49	H-NS	48	92	2294	2491
pSG1	NC_007182	Sodalis glossinidius	81553	49		48	92	56217	56414
pSMS35_130	NC_010488	Escherichia coli SMS-3-5	130440	51		55	92	3364	3167	MOBF
pSS_046	NC_007385	Shigella sonnei Ss046	214396	45		55	92	178363	178560	MOBF
pUTI89	NC_007941	Escherichia coli UTI89	114230	51		53	92	113993	114190	MOBF
pWR501	NC_002698	Shigella flexneri	221851	46		55	92	207534	207731	MOBF
R100	NC_002134	Shigella flexneri 2b str. 222	94281	52		53	92	87185	87382	MOBF
R27	NC_002305	Salmonella enterica subsp. enterica serovar Typhi	180461	46	H-NS	47	100	159402	159196	MOBH
R478	NC_005211	Serratia marcescens	274762	46	H-NS	50	92	59426	59623	MOBH
R721	NC_002525	Escherichia coli	75582	43		66	90	35285	35091
Unnamed	NC_011148	Salmonella enterica subsp. enterica serovar Agona str. SL483	37978	41	H-NS	42	93	1363	1163

aThis 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%) (Figure 2(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.

Figure 2

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.

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 name	Accession no.	Source organism	Length (nt)	G+C content (%)b	NAP gene homolog	Identity (%)c	Query coverage (%)	Subject start	Subject end
1	NC_007949	Polaromonas sp. JS666	360405	57	HU	52	99	61052	60786
1	NC_008573	Shewanella sp. ANA-3	278942	46
2	NC_007950	Polaromonas sp. JS666	338007	60
ICEhin1056	NC_011409	Haemophilus influenzae	59393	39
pAM04528	NC_012693	Salmonella enterica	158213	52	HU	57	99	14067	14333
pAPEC-O1-R	NC_009838	Escherichia coli APEC O1	241387	46
pAR060302	NC_012692	Escherichia coli	166530	53	HU	57	99	15755	16021
pAsa4	NC_009349	Aeromonas salmonicida subsp. salmonicida A449	166749	53	HU	60	99	26844	26578
pCAR1	NC_004444	Pseudomonas resinovorans	199035	56	MvaT	61	98	77640	77993
					NdpA	36	98	95390	94395
					HU	42	99	97809	98075
pEC-IMP	NC_012555	Enterobacter cloacae	318782	48	H-NS	64	99	109370	108969
pEC-IMPQ	NC_012556	Enterobacter cloacae	324503	48	H-NS	64	99	109370	108969
peH4H	NC_012690	Escherichia coli	148105	53	HU	57	99	14067	14333
pHCM1	NC_003384	Salmonella enterica subsp. enterica serovar Typhi str. CT18	218160	48	H-NS	61	99	131861	131460
pIP1202	NC_009141	Yersinia pestis bv. Orientalis str. IP275	182913	53	HU	57	99	14067	14333
pK29	NC_010870	Klebsiella pneumoniae	269674	46
plasmid1	NC_007901	Rhodoferax ferrireducens T118	257447	54
pMAK1	NC_009981	Salmonella enterica subsp. enterica serovar Choleraesuis	208409	47	H-NS	61	99	60046	59645
pMAQU02	NC_008739	Marinobacter aquaeolei VT8	213290	53
pO111_1	NC_013365	Escherichia coli O111:H- str. 11128	204604	47	H-NS	61	99	80175	79774
pP91278	NC_008613	Photobacterium damselae subsp. Piscicida	131520	52	HU	57	99	125918	126184
pP99-018	NC_008612	Photobacterium damselae subsp. piscicida	150157	51	HU	57	99	133314	133580
pRA1	NC_012885	Aeromonas hydrophila	143963	51	HU	58	99	15573	15839
pRp12D01	NC_012855	Ralstonia pickettii 12D	389779	58	HU	37	99	321346	321080
pSN254	NC_009140	Salmonella enterica subsp. enterica serovar Newport str. SL254	176473	53	HU	57	99	14067	14333
pTK9001	NC_013930	Thioalkalivibrio sp. K90mix	240256	62
pYR1	NC_009139	Yersinia ruckeri	158038	51	HU	57	99	15070	15336
R27	NC_002305	Salmonella enterica subsp. enterica serovar Typhi	180461	46	H-NS	61	99	148225	148626
R478	NC_005211	Serratia marcescens	274762	46	H-NS	64	99	111747	111346
Rts1	NC_003905	Proteus vulgaris	217182	46

aThis 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 name	Accession no.	Source organism	Length (nt)	G+C content (%)b	NAP gene homolog	Identity (%)c	Query coverage (%)	Subject start	Subject end
1	NC_008242	Chelativorans sp. BNC1	343931	62	HU	41	94	133932	133678
3	NC_007617	Nitrosospira multiformis ATCC 25196	14159	50
3	NC_007961	Nitrobacter hamburgensis X14	121408	62
At	NC_003064	Agrobacterium tumefaciens str. C58	542868	57	IHF	36	82	112654	112412
C	NC_010542	Cyanothece sp. ATCC 51142	14685	38
ColE9-J	NC_011977	Escherichia coli	7577	50
DN1	NC_002636	Dichelobacter nodosus	5112	62
F plasmid	NC_008036	Sphingopyxis alaskensis RB2256	28543	60
p11745	NC_013546	Actinobacillus pleuropneumoniae	5486	38
p12494	NC_010889	Actinobacillus pleuropneumoniae	14393	33
p1ABAYE	NC_010401	Acinetobacter baumannii AYE	5644	35
p1META1	NC_012807	Methylobacterium extorquens AM1	44195	68
p1METDI	NC_012987	Methylobacterium extorquens DM4	141504	65
p2007057	NC_011897	Salmonella enterica subsp. enterica serovar Bovismorbificans	4270	47
p2ABSDF	NC_010396	Acinetobacter baumannii SDF	25014	35
p2ESCUM	NC_011739	Escherichia coli UMN026	33809	42
p2META1	NC_012809	Methylobacterium extorquens AM1	37858	65	IHF	44	95	28369	28635
p3ABSDF	NC_010398	Acinetobacter baumannii SDF	24922	34
p42a	NC_007762	Rhizobium etli CFN 42	194229	58
p49879.1	NC_006907	Leptospirillum ferrooxidans	28878	58	HU	47	99	3281	3015
p49879.2	NC_006909	Leptospirillum ferrooxidans	28012	55	HU	48	99	15858	15592
pAb5S9	NC_009476	Aeromonas bestiarum	24716	54
pACRY07	NC_009473	Acidiphilium cryptum JF-5	5629	58
pAgK84	NC_011994	Agrobacterium radiobacter K84	44420	54
pAM5	NC_008691	Acidiphilium multivorum	5161	58
pAMI2	NC_010847	Paracoccus aminophilus	18563	62
pAMI3	NC_013513	Paracoccus aminophilus	5575	61
pAPA01-030	NC_013212	Acetobacter pasteurianus IFO 3283-01	49961	54
pAPA01-040	NC_013213	Acetobacter pasteurianus IFO 3283-01	3204	54
pAtK84b	NC_011990	Agrobacterium radiobacter K84	184668	59	IHF	38	86	54109	53855
pAtS4b	NC_011991	Agrobacterium vitis S4	130435	56	IHF	47	97	44880	45152
pAtS4c	NC_011984	Agrobacterium vitis S4	211620	59	HU	45	94	141245	140991
pAtS4e	NC_011981	Agrobacterium vitis S4	631775	57	HU	41	94	40476	40222
					Lrp	30	87	460443	460871
					Lrp	34	74	425247	424888
pAV2	NC_010310	Acinetobacter venetianus	15135	36
pB	NC_010996	Rhizobium etli CIAT 652	429111	58
pBGR3	NC_012847	Bartonella grahamii as4aup	28192	36
pBS512_5	NC_010659	Shigella boydii CDC 3083-94	5114	46
pC	NC_010997	Rhizobium etli CIAT 652	1091523	61	Lrp	46	88	617696	618130
					Lrp	42	90	609059	608619
					Lrp	39	95	417738	418202
					Lrp	42	79	714804	715193
					Lrp	39	93	406570	407025
pCAUL01	NC_010335	Caulobacter sp. K31	233649	67	HU	44	99	97598	97329
					Lrp	34	89	182479	182042
pCAUL02	NC_010333	Caulobacter sp. K31	177878	64
pCCK1900	NC_011378	Pasteurella multocida	10226	61
pCCK381	NC_006994	Pasteurella multocida	10874	61
pCFPG4	NC_011563	Candidatus Azobacteroides pseudotrichonympha genomovar. CFP2	4149	44
pCHE-A	NC_012006	Enterobacter cloacae	7560	60
pColE8	NC_012882	Escherichia coli	6751	51
pCROD3	NC_013719	Citrobacter rodentium ICC168	3910	51
pCT02021853_74	NC_011204	Salmonella enterica subsp. enterica serovar Dublin str. CT_02021853	74551	49
pCVM19633_4	NC_011093	Salmonella enterica subsp. enterica serovar Schwarzengrund str. CVM19633	4585	48
pDSHI01	NC_009955	Dinoroseobacter shibae DFL 12	190506	60
pET09	NC_010695	Erwinia tasmaniensis Et1/99	9299	47
pGDIA01	NC_011367	Gluconacetobacter diazotrophicus PAl 5	27455	59
pGOX3	NC_006674	Gluconobacter oxydans 621H	14547	56
pHCG3	NC_005873	Oligotropha carboxidovorans OM5	133058	61
pHRM2a	NC_012109	Desulfobacterium autotrophicum HRM2	68709	42
pIGJC156	NC_009781	Escherichia coli	5146	47
pIGMS5	NC_010883	Escherichia coli	6750	51
pIGWZ12	NC_010885	Escherichia coli	4072	50
pISP3	NC_013970	Sphingomonas sp. MM-1	43398	63
pJD4	NC_002098	Neisseria gonorrhoeae	7426	38
plasmid1	NC_007801	Jannaschia sp. CCS1	86072	58
pLD-TEX-KL	NC_009966	Fluoribacter dumoffii	66512	39
pMAC	NC_006877	Acinetobacter baumannii	9540	35
pMAS2027	NC_013503	Escherichia coli	42644	43
pMbo4.6	NC_013500	Moraxella bovis	4658	39
pMCHL01	NC_011758	Methylobacterium chloromethanicum CM4	380207	66
pMG160	NC_004527	Rhodobacter blasticus	3431	67
pMG828-2	NC_008487	Escherichia coli	4091	50
pMG828-4	NC_008489	Escherichia coli	7462	48
pMMCU1	NC_013056	Acinetobacter calcoaceticus	8771	35
pMMCU2	NC_013506	Acinetobacter baumannii	10270	36
pMRAD01	NC_010510	Methylobacterium radiotolerans JCM 2831	586164	70
pMS260	NC_005312	Actinobacillus pleuropneumoniae	8124	61
pNGR234a	NC_000914	Rhizobium sp. NGR234	536165	59	Lrp	41	70	197189	196845
					Lrp	30	89	188867	188430
pNGR234b	NC_012586	Rhizobium sp. NGR234	2430033	62	Lrp	46	90	656547	656107
					Lrp	45	85	667494	667913
					Lrp	43	90	1038020	1038463
					Lrp	44	85	682796	683215
					Lrp	38	96	2400849	2401319
					Lrp	44	79	709104	708715
					Lrp	41	89	28336	28761
					Lrp	33	89	1108900	1109337
					Lrp	36	90	703213	702764
					Lrp	32	77	1112953	1112582
pNL2	NC_009427	Novosphingobium aromaticivorans DSM 12444	487268	66
pO111_4	NC_013367	Escherichia coli O111:H- str. 11128	8140	50
pO26-S4	NC_011228	Escherichia coli	6758	51
pOLA52	NC_010378	Escherichia coli	51602	46
pOU1114	NC_010421	Salmonella enterica subsp. enterica serovar Dublin	34595	42
pOU1115	NC_010422	Salmonella enterica subsp. enterica serovar Dublin	74589	49
pP	NC_003455	Salmonella enterica subsp. enterica serovar Enteritidis	4301	50
pP742405	NC_011733	Cyanothece sp. PCC 7424	18083	38
pP742406	NC_011734	Cyanothece sp. PCC 7424	15219	40
pPMA4326C	NC_005921	Pseudomonas syringae pv. maculicola	8244	53
pPNAP07	NC_008763	Polaromonas naphthalenivorans CJ2	9898	57
pPRO2	NC_008608	Pelobacter propionicus DSM 2379	30722	56
pPT1	NC_002143	Comamonas testosteroni	15398	56
pR132501	NC_012848	Rhizobium leguminosarum bv. trifolii WSM1325	828924	60	Lrp	47	88	234905	234471
					Lrp	44	86	386338	386760
					Lrp	39	93	645542	645087
					Lrp	42	79	147165	146776
pR132502	NC_012858	Rhizobium leguminosarum bv. trifolii WSM1325	660973	61
pR132503	NC_012853	Rhizobium leguminosarum bv. trifolii WSM1325	516088	59	HU	47	94	300662	300916
pR132504	NC_012852	Rhizobium leguminosarum bv. trifolii WSM1325	350312	61
pR132505	NC_012854	Rhizobium leguminosarum bv. trifolii WSM1325	294782	60
pRF	NC_007110	Rickettsia felis URRWXCal2	62829	34
pRFdelta	NC_007111	Rickettsia felis URRWXCal2	39263	33
pRi1724	NC_002575	Agrobacterium rhizogenes	217594	57
pRi2659	NC_010841	Agrobacterium rhizogenes	185462	58
pRL10	NC_008381	Rhizobium leguminosarum bv. viciae 3841	488135	60
pRL11	NC_008384	Rhizobium leguminosarum bv. viciae 3841	684202	61
pRL12	NC_008378	Rhizobium leguminosarum bv. viciae 3841	870021	61	Lrp	46	88	599116	598682
					Lrp	43	88	658287	658718
					Lrp	39	93	45601	45146
					Lrp	42	79	450080	449691
pRL7	NC_008382	Rhizobium leguminosarum bv. viciae 3841	151564	58	HU	48	94	20484	20230
pRL8	NC_008383	Rhizobium leguminosarum bv. viciae 3841	147463	59	Lrp	33	87	70763	70344
pRLG201	NC_011368	Rhizobium leguminosarum bv. trifolii WSM2304	1266105	60	Lrp	45	89	917573	917136
					Lrp	44	85	41998	42417
					Lrp	44	79	473039	472650
					Lrp	40	93	1162146	1161691
					Lrp	40	93	1150939	1150484
					Lrp	32	88	707587	707162
pRM	NC_010927	Rickettsia monacensis	23486	32
pSC101	NC_002056	Salmonella enterica subsp. enterica serovar Typhimurium	9263	51
pSE11-6	NC_011411	Escherichia coli SE11	4082	49
pSE34	NC_010860	Salmonella enterica subsp. enterica serovar Enteritidis	32950	41
pSMED01	NC_009620	Sinorhizobium medicae WSM419	1570951	62	Lrp	40	77	143180	143557
					Lrp	34	89	574284	573847
pSMED02	NC_009621	Sinorhizobium medicae WSM419	1245408	60	Lrp	42	91	556486	556932
					Lrp	40	91	842324	842758
					Lrp	31	87	22345	21917
pSmeSM11a	NC_013545	Sinorhizobium meliloti	144170	60	Lrp	46	96	70449	70922
pSmeSM11b	NC_010865	Sinorhizobium meliloti SM11	181251	59
pSMS35_4	NC_010486	Escherichia coli SMS-3-5	4074	50
pSx-Qyy	NC_006826	Sphingobium xenophagum	5683	56
pSymA	NC_003037	Sinorhizobium meliloti 1021	1354226	60	Lrp
pSymB	NC_003078	Sinorhizobium meliloti 1021	1683333	62	Lrp	38	90	440778	440335
					Lrp	36	89	29555	29992
pTB3	NC_008388	Roseobacter denitrificans OCh 114	16575	55
pTcM1	NC_010600	Acidithiobacillus caldus	65158	57	IHF	56	89	25186	25449
pTiS4	NC_011982	Agrobacterium vitis S4	258824	57	HU	41	94	27356	27102
					HU	40	94	83408	83154
					Lrp	42	79	96920	97309
pTi-SAKURA	NC_002147	Agrobacterium tumefaciens	206479	56	HU	44	94	95763	95509
pUT1	NC_014005	Sphingobium japonicum UT26S	31776	64
pUT2	NC_014009	Sphingobium japonicum UT26S	5398	61
pXAUT01	NC_009717	Xanthobacter autotrophicus Py2	316164	65
pXCV19	NC_007505	Xanthomonas campestris pv. vesicatoria str. 85-10	19146	60
pXF51	NC_002490	Xylella fastidiosa 9a5c	51158	50
pYAN-1	NC_008246	Sphingobium yanoikuyae	5182	62
pYAN-2	NC_008247	Sphingobium yanoikuyae	4924	64
RSF1010	NC_001740	Escherichia coli	8684	61
Symbiotic plasmid p42d	NC_004041	Rhizobium etli CFN 42	371254	58
Ti	NC_002377	Agrobacterium tumefaciens	194140	55	IHF	43	97	180164	180436
Ti	NC_003065	Agrobacterium tumefaciens str. C58	214233	57	HU	44	94	139735	139481
Ti plasmid pTiBo542	NC_010929	Agrobacterium tumefaciens	244978	55	IHF	36	86	209743	209489
					IHF	45	98	187204	187479
Unnamed	NC_011143	Phenylobacterium zucineum HLK1	382976	69

aThis 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.