Open Journal of Genetics, 2012, 2, 5-10 OJGen
Published Online December 2012 (http://www.SciRP.org/journal/ojgen/)
Published Online March 2012 in SciRes. http://www.scirp.org/journal/ojgen
Organization, expression and evolution of flagellar genesin
Rhodobacter sphaeroides 2.4.1
Durga Thapaliya1,B. Myagmarjav1, C. Trahan1,D. Ortiz1,H. Cho2, M. Choudhary1*
1Department of Biological Sciences, Sam Houston State University, Huntsville, Texas, USA
2Department of Computer Science, Sam Houston State University, Huntsville, Texas, USA
Received 2012
ABSTRACT
Rhodobacter sphaeroides 2.4.1 belongs to theα-3 sub-
division of the Proteobacteria. It possesses a multipar-
tite genome structure consisting of two circular
chromosomes, andit displays a wide range of meta-
bolic diversity.Approximately 40 flagellar proteins
are required for structure, assembly, and regulation
of the flagellum formation in most bacterial species. R.
sphaeroidescontains two flagellar gene clusters (fla1
and fla2),which encode 38 and 21 proteins, respec-
tively. Thirty-six of these genes exist in duplicate
gene-pairs.A combination of genome analysis, phylo-
genetic analysis and mRNA expression analysis were
employed to examine the conservation of structure,
function and evolution of fla1 and fla2 in R. sphae-
roides. The results demonstrated that fla2, which was
shared among members of α-Proteobacteria, is native
toR. sphaeroides, while fla1 was horizontally trans-
ferred from a member of γ-Proteobacteria.In addition,
genes located in fla1 are expressed over several
growth conditions, but those in fla2 are barely ex-
presse d.
Keywords: Flagella; Horizontal Gene Transfer;
Phylogenetic Tree
1. INTRODUCTION
Bacterial flagella are complex structures that facilitate
different types of motilities (swimming, swarming, gliding
and twitching), and play important roles in sensing outside
environments (temperature, nutrient and oxygen
availability), adhesion, biofilm production, and host
invasion [1]. A bacterial flagellum is composed of at least
21-24 core proteins [2, 3], which represent six structural
components of the flagellum, including a basal body (MS
ring, P ring, and L ring), a motor, a switch, a hook, a
filament, and an export apparatus.In addition, another set of
15-25 proteins is responsible for the regulation of flagellar
assembly and the uncovering and processing of
environmental signals to which flagella respond [4].
A large number of bacterial species contain a single
flagellar gene cluster, which providescells with different
types of motilities. However, a number of bacterial species
inthe generaVibrio,Rhodospirillum, Bradyrhizobium,
Burkholderia, and Yersiniapossess twoflagellar gene
clusters [5], which contain genes that encode proteins for
the synthesis ofpolar and lateral flagella to control
swimming and swarming, respectively. Although the ability
to synthesize different flagella types is primarily encoded
by structural genes, the variation in regulation mechanisms
providesthese microorganisms varied strategies to exploit a
diverse range of ecological niches.
R. sphaeroides is a purple non-sulfur photo synt hetic
bacterium, which belongs to the α-3 subgroup of
theProteobacteria [6].The genome of R. sphaeroides
consists of two circular chromosomes [7], which has
been completely sequenced and annotated [8]. It also
exhibits a prevalence of gene duplications [9, 10].
Genome analysis revealed that the primary
chromosome of R. sphaeroides contains two flagellar
gene clusters, fla1 (between 1,736,242 and 1,951,757
base-pairs) and fla2 (between 3,074,540 and 3,105,787
base-pairs). It has been found that the fla1 cluster
contains 38genes, and is responsible for the formation
ofthe polar flagellum, while fla2 cluster contains 21
genes, whose functions remain unclear.
The duplicate gene-pairs that exist between fla1 and
fla2 clusters in R. sphaeroidesmay have resulted from
either segmental gene duplicationor horizontal gene
transfer [11]. The two gene clusters may have diverged
since gene duplication or horizontal gene transfer,
andthe two gene clusters would have evolved and
expressed differently under different growth conditions.
To study structure and function of these duplicate
flagellargenes, current study employsthe following
approaches, includingsequence analysis, phylogenetic
*Corresponding author.

D. Thapaliya et al. / Open Journal of Genetics 2 (2012) 5-10
analysis, and mRNA expression analysis.
Table1.Similarity of duplicate genes between fla1 and fla2
clusters in R. sphaeroides 2.4.1.
Ge ne
a
RSP No.
b
Cov erage
c
Score
d
Identity
e
E-va l uef
f lhA 1 / flhA2 0034 / 1320 97 364 36 3.00E-117
flgI1 / flgI2 0076 / 1307 93 249 43 8.00E-81
flgG1 / flgG2 0078 / 1326 96 207 42 1.00E-67
fliP1 / fliP2 0063 / 1309 90 176 40 2.00E-55
flhB1 / flhB2 0066 / 1322 94 136 33 2.00E-38
flgE1 / flgE2 0080 / 1303 98 108 25 4.00E-28
fliF1 / fliF2
0053 / 1312
77
103
30
1.00E-25
fliR1 / fliR2
0065 / 1321
85
77
33
8.00E-19
flgF1 / flgF2
0079 / 1327
90
77
32
4.00E-19
flgH1 / flgH2 0077 / 1324 74 75.1 30 7.00E-19
fliQ1 / fliQ2 0064 / 1328 79 62.8 46 3.00E-16
flgD1 / flgD2 0081 / 1336 30 62.4 46 3.00E-14
flgK1 / flgK2 0074 / 1304 64 46.6 37 3.00E-07
motA1 / motA2 0233 / 1316 87 43.5 22 1.00E-07
flgA1 / flgA2 0036 / 1325 70 43.1 33 8.00E-08
flgC1 / flgC2 0082 / 1330 83 41.2 43 7.00E-08
flgB1 / flgB2
0083 / 1331
71
40
32
2.00E-07
fliE1 / fliE2
0052 / 1329
100
32
28
1.00E-04
aGene name reflecting cluster 1 (fla1) and 2 (fla2),bR. sphaeroides gene num-
ber,cPercentage query coverage,dNorma lized score,ePercentage amino acid
identity,fExpected value
2. MATERIALS AND METHODS
2.1. Sequence Analysis
Identification of a gene homolog within the genome of
R. sphaeroideswas performed using gapped BLASTP
[12], where genes from fla1 were used as queries to
identify the corresponding homolog in the fla2 cluster.
Each copy of the duplicate gene-pair was also used to
identifyorthologs among bacterial species representing
different groups of Proteobacteria, using the
symmetrical best-hit method. To identify the orthologs,
three criteria (E-score threshold <10-3, query coverage
of 50%, and overall amino acid identity >30%) were
used. All duplicate genes were analyzed for their %GC
content, di- and tri-nucleotide repeat patterns, and
codon usage.
2.2. Global DNA Sequence Alignment
The flagellar gene clusters were identified in the
genomic sequences of R. sphaeroides 2.4.1,
Ruegeriapomeroyi DSS-3, and
Pseudoxanthomonassuwonensis 11-1 . DNA sequence
files (in fasta format)of each flagellar cluster were
downloaded from the NCBI database and the sequence
alignments were performed using Mauve 2.3.1 [13].
Number of locally collinear blocks (LCBs) and %
conservation of the DNA regionsweredetermined as
previously described [9].
2.3. Phylogenetic Analysis
Phyloge ne tic analysis was performed for the five
coregenes (flhA, flgG, fliP,flhB, and flgE),which were
selected with the criteria of percentage query
coverage >90% and normalized score >100. Geneious
v4.6 was used to generate alignments using MUSCLE
[14] and construct phylogenetic treesusing
Neighb or-Joining (NJ) [15] and Maximum-Likelihood
(ML)methods [16]with 100 bootstrap replications.
Protein sequences of these corresponding genes were
obtained from a total of 75 proteobacterial species
(15α-, 15 β-, 28 γ-, 8 δ-, and 9 ε-) for phylogenetic tree
const ruct ion.
2.4. Expression Analysis
Microarray expression data ofR. sphaeroides2.4.1 were
available [17], from which the expression data of the
flagellar genes were collected and used for this study.
The expression levels were measured under seven
growth conditions, includingthree photosynthetic
conditions (3 watts, 10 watts, and 100 watts), aerobic,
semi -aerobic, and dark-dimethyl-sulfoxide(DMSO).
Individual and average expression levels of genes in
fla1 and fla2 were compared.
3. RESULTS
3.1. Identification of Flagellar Gene Homo-
logs in R. sphaeroides genome
A majority offlagellar genes are located in the two gene
clusters, fla1 (between 1,736,242 and 1,951,757
base-pairs) and fla2 (between 3,074,540 and 3,105,787
base-pairs) on the primary chromosome of R. sphaer-
oides as previously designated [11].The fla1 and fla2
gene clusters encode 38 and 21 proteins, respectively. A
singleton gene (fliG2) is located in a different region
(between 831,570 and 832,643 base-pairs) of the same
chromosome. In addition, a mini-cluster containing four
genes (flaA, fla F, flb T, and flg F) are located in plasmid
pRS241A. The resultsof the amino acid similarity be-
tween 18 gene-pairs wereshown in Table 1.T he amino
acid identities for these gene-pairs range from 22% to
Table 2.Comparison of genomic alignments of the flagellar
gene clusters.
Aligned flagellar clusters No.LC
Bsa
LCBL
engthb
%
Conservedc
R.sphaeroides-fla1(α)/R.sphaeroides-fla2(
α) 23 41065 13.02
R. sphaeroides-fla1(α) / R. pomeroyi(α) 18 34693 8.38
R. sphaeroides-fla1(α) / P. suwonensis(γ) 21 68487 16.08
R. sphaeroides-fla2(α) / R. pomeroyi(α) 5 44714 25.78
R. sphaeroides-fla2(α) / P. suwonensis(γ) 11 41542 8.01
R.pomeroyi(α) / P. suwonensis(γ) 19 31978 16.07
alocalcolinear block (LCB), bLCB length (in base-pair), cLCB percentage (over the
total length of gene cluster)
6

D. Thapaliya et al. / Open Journal of Genetics 2 (2012) 5-10
46%. Six of the 18 gene-pairs exhibit similarity >90% of
their corresponding protein lengths with >100 normal-
ized scores. Furthermore, the genes located in the
fla1 cluster also indicated significant matches to the
members of the γ-Proteobacteria, and the amino acid
identity between the two copies of each corresponding
ortholog were higher than the amino acid identity shown
between the corresponding duplicate copies withinR.
spha er oides. In contrast, genes in the fla2 cluster mostly
match to the corresponding homologs
ofα-proteobacterial species (data not shown).
3.2. Comparison of fla1 and fla2
Comparisons of all pairwise DNA sequence alignments
of the fla1 and fla2 regions of R. sphaeroides, R.
pomeroyi, and P. suwonensis were described in Table 2.
Thealignments between fla1and fla2 of R. sphaeroides,
between fla1 of R. sphaeroides and the flagellar region
of P. suwonensis, and between fla2 of R. sphaeroides
and the flagellar region ofR. pomeroyi were shown in
Figure 1A, 1B, and 1C, respectively. As shown in
Table 2, fla2 of R. sphaeroides and the flagellar gene
cluster of R.pomero yishared five LCBs with 25.78%
sequence conservation.The organization of LCBs in
thegenomic regionis indicative of two large scale
inversions in R. pomeroyi as shown in Figure 1C.
However, fla1 and fla2 of R. sphaeroides shared 23
LCBs with the low level (13.02%)of their sequence
conservation, which is also demonstrated by small
LCBs with a large number of chromosomal
rearrangements as shown in Figure 1A. The fla1 cluster
of R. sphaeroidesand the flagellar cluster of
P. suwonensis11 -1 shared 21LCBs with 16.07%
sequence conservation, and this medium level
conservation is also reflected in larger LCBs as shown
in Figure 1B.
The five common genes in fla1 and fla2 clusters of R.
sphaeroides, and their corresponding homologs in R.
pomeroyi and P. suwonensis were analyzed for the %GC
content, di- and tri-nucleotide repeats, and codon
frequencies. The % GC content, di- and tri-nucleotide
repeat, and codon frequency distributions are similar
among these gene homologs (data not shown).
3.3. Phylogenetic Analysis
P hyl ogenetic analysis based on each of the fivepro tein
(FlhA, FlgG, FliP, FlhB, and FlgE)trees reflected a si mi-
lar evolutionary relationship among 75 proteobacterial
species. Two FlgG trees based on NJ distance and max-
imu m-likelihood methodswere shown in Figure 2A and
2B, respectively. The results revealed that genes located
in fla2 of R. sphaeroides form a clade with its closely
related species, R. pomeroyi and P. denitrificans with a
bootstrap value of 100. All three species belong to the
order Rhodobacteriales, and are located within the
branch of α-Proteobacteria. In contrast, the genes lo-
Figure 1.Mauve representation of flagellar clusters of R. sphaeroides 2.4.1, P. suwonensis, and R. pomeroyi. Each se-
quential color-block represents a homologous backbone DNA sequence without rearrangement.
7