E---40-2--10-166-07180.mdiActa Biochim Biophys Sin (2008) | Volume
40 | Issue 2 | Page 166
Sinorhizobium meliloti lrhA subfamily genesActa Biochim Biophys Sin
(2008): 166-173 | © 2008 Institute of Biochemistry and Cell
Biology, SIBS, CAS | All Rights Reserved 1672-9145
http://www.abbs.info; www.blackwellpublishing.com/abbs | DOI:
10.1111/j.1745-7270.2008.00378.x
Characteristics of the LrhA subfamily of transcriptional regulators
from Sinorhizobium meliloti
Mingsheng Qi1,2#, Li Luo1,2#, Haiping Cheng3, Jiabi Zhu1, and
Guanqiao Yu1* 1 Laboratory of Molecular Genetics, Shanghai
Institute of Plant Physiology and Ecology, Shanghai Institute for
Biological Sciences,
Chinese Academy of Sciences, Shanghai 200032, China 2 Graduate
School of the Chinese Academy of Science s, Beijing 100049, China 3
Biological Sciences Department, Lehman College, City University of
New York, New York 10468, USA
Received: June 20, 2007 Accepted: October 29, 2007 This work was
supported by the grants from National Key Program for Basic
Research of China (No. 2001CB108901) # These authors contributed
equally to this work *Corresponding author: Tel, 86-21-54924165;
Fax, 86-21-54924015; E-mail,
[email protected]
In our previous work, we identified 94 putative genes encod- ing
LysR-type transcriptional regulators from Sinorhizobium meliloti.
All of these putative lysR genes were mutagenized using plasmid
insertions to determine their phenotypes. Six LysR-type regulators,
encoded by mutants SMa1979, SMb20715, SMc00820, SMc04163, SMc03975,
and SMc04315, showed similar amino acid sequences (30%) and shared
the conserved DNA-binding domain with LrhA, HexA, or DgdR.
Phenotype analysis of these gene mutants indicated that the
regulators control the swimming behaviors of the bacteria,
production of quorum-sensing signals, and secretion of
extracellular proteins. These characteristics are very similar to
those of LrhA, HexA, and DgdR. Thus, we refer to this group as the
LrhA subfamily. Sequence analysis showed that a great number of
homologous genes of the LrhA subfamily were distributed in the α,
β, and γ subdi- visions of proteobacteria, and a few in
actinobacteria. These findings could provide new clues to the roles
of the LysR gene family.
Keywords LrhA subfamily; LysR-type transcriptional regulator;
Sinorhizobium meliloti
The LysR family of regulators, evolved from distant ancestors, are
broadly distributed in prokaryotic genera. The structure and
function of the LysR family of tran- scriptional regulators are
conserved to some extent. They are typically approximately 300
amino acids long with an
N-terminal DNA-binding domain participating in the recognition of
target promoter, and a C-terminal domain for sensing signal
molecules [1]. They function as transcriptional activators or
repressors. Typically they regulate genes with promoters different
from their own. The promoters of the target genes often have a
conserved sequence and typically at least one TN11A motif [1]. The
conserved and divergently oriented promoters of target genes to
lysR regulatory genes can facilitate the quick recognition of these
promoters for us.
One of the LysR-type regulator genes, lrhA from Escherichia coli,
is located upstream of the nuoA-N (NADH:quinone oxidoreductase)
locus [2]. LrhA mainly functions in controlling the transcription
of flagella, motility, and chemotaxis genes by regulating the
expression of the flhDC regulon, the master regulator of flagella-
and motil- ity-related genes [3]. The LrhA protein is highly
homolo- gous to HexA from Erwinia carotovora (64% identity) and
PecT from Erwinia chrysanthemi (61% identity). In some
phytopathogenic bacteria, HexA and PecT act as motility repressors
and virulence factors, such as exoenzymes re- quired for lytic
reactions [4,5]. Overexpression of the Erw. carotovora hexA gene in
the opportunistic human patho- gen Serratia also represses multiple
virulence determinants [5]. In hexA mutants of Erw. carotovora,
expression of flagella genes (fliA and fliC) is increased, thereby
result- ing in hypermotility [5]. In the same organism, HexA also
regulates the production of the regulatory RNA rsmB (a homolog of
the E. coli csrB), the quorum-sensing phero- mone
N-(3-oxohexanoyl)-L-homoserine lactone, and the stationary phase
sigma factor RpoS [6].
In our previous work [7], 94 putative LysR family genes were
mutagenized by the insertion of suicide plasmids. Phenotype
determination of these mutants indicated that mutation of six genes
among them impaired the motility of
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Sinorhizobium meliloti lrhA subfamily genes
the strains in rich medium. The products encoded by these six genes
are highly homologous with LrhA and HexA; they belong to the same
clade, as revealed by the phylog- eny analysis of 90 putative LysR
family genes. Referring to HexA, several other experiments were
also carried out, such as homoserine lactone assay and
quantification of extracellular protein. Three mutants, Sm326,
Sm341, and Sm360 excreted less N-acyl homoserine lactone (AHL) than
the wild-type Rm1021, whereas the other three mu- tants had no
difference from the wild type. Only Sm326 secreted more
extracellular proteins than the wild type. These findings suggested
that these six LysR-type regula- tors have sequences and functions
similar to those of LrhA and HexA. In particular, product encoded
by SMa1979 showed functions in regulating cell motility, AHL
production, and extracellular protein secretion similar to those of
Erw. carotovora HexA. We refer to this group as the LrhA
subfamily.
Therefore, homologs of LrhA genes from sequenced bacterial genomes
were collected to find significantly different distributions in
those bacteria by analyzing their genomic sequences.
Materials and Methods
Bacterial strains and medium The bacterial strains used in this
work are listed in Table
1. Luria-Bertani (LB) medium was used for the growth of E. coli.
The ZMGS (10 g/L mannitol, 1 g/L glutamic acid, 1 g/L K2PO4, 1
mg/ml MnCl2, 0.1 mg/ml H3BO3, 0.1 mg/ ml ZnSO4·7H2O, 0.1 mg/ml
CoCl2·6H2O, 0.1 mg/ml CuSO4·5H2O, 10 mg/ml FeCl3, 1 mg/ml biotin,
and 1 mg/ ml thiamine) and LB media used for Sinorhizobium meliloti
were supplemented with 2.5 mM MgSO4 and 2.5 mM CaCl2 (LB/MC). Agar
(1.5%) was used as the solid media. Antibiotics were used at the
following concentrations: kanamycin, 25 µg/ml; ampicillin, 100
µg/ml; neomycin, 200 µg/ml; streptomycin, 500 µg/ml; tetracycline,
10 µg/ ml; spectinomycin, 100 µg/ml; and gentamicin, 50
µg/ml.
Motility test Cell motility was examined using both microscopy and
medium for swimming as described previously by Wei and Bauer [13].
Briefly, bacterial strains were inoculated onto LB/MC and ZMGS soft
agar media (0.3%) and incu- bated for 4 d to determine their colony
size. Photographs were taken using a Nikon Coolpix 4500 digital
camera (Nikon, Tokyo, Japan).
AHL bioassay AHL assays were carried out as reported by Marketon
and González [14] with some modifications. Briefly, 150 µl
supernatant of bacteria culture was mixed with 30 µl indi- cator
strain Agrobacterium tumefaciens NTL4 (pZLR4)
Table 1 Strains of bacteria used in this work
Bacterial strains or plasmids Relevant characteristics Reference or
source
Sinorhizobium meliloti Rm1021 Strr, derivative of SU47 [8] Sm326
SMa1979::pK19mob2HMB [7] Sm341 SMb20715::pK19mob2HMB [7] Sm360
SMc00820::pK19mob2HMB [7] Sm379 SMc04163::pK19mob2HMB [7] Sm382
SMc03975::pK19mob2HMB [7] Sm383 SMc04315::pK19mob2HMB [7]
Agrobacterium tumefaciens NTL4 (pZLR4) Derivative of NT1 carrying a
traG::lacZ reporter fusion Agrr, Gmr [9,10] Escherichia coli DH5α
SupE44lacU169(80lacZ15)hsdR17recA1endA1 Laboratory stock MT616
Pro-82thi-1endA1supE44−,Cmr, carrying pRK600 [11] Plasmids pAtC58
Cryptic plasmid of A. tumefaciens [10] pTiC58accR Derivative of
pTiC58 with a deletion in the accR gene, Trac [12]
Agrr, agrocin 84 resistance; AHL, N-acyl-homoserine lactone; Cmr,
chloramphenicol resistance; Gmr, gentamicin resistance; Strr,
streptomycin resistance; Tcs, tetracycline sensitive; Trac,
transfer constitutive.
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Sinorhizobium meliloti lrhA subfamily genes
[9]. NTL4 (pAtC58) and NTL4 (pAtC58, pTiC58accR) were used as the
negative and positive controls, respec- tively [10,15]. The
relative amount of AHL of those bacte- ria was determined by
measuring the β-galactosidase ac- tivity of the indicator strain
after 3 h. The β-galactosidase assays were carried out as described
by Miller [16]. Spec- trophotometer 7200 (Tianmei Scientific
Equipment Cooperation, Shanghai, China) was used in this
work.
Total extracellular protein assays Quantitative spectrophotometric
assays were carried out to assess the total extracellular proteins
produced by the wild type and mutants of the LrhA subfamily
regulator when OD600 nm is 0.2, 2.0, and 5.0, using the Coomassie
Brilliant Blue G-250 method described by Bradford [14].
Multiple sequence alignment The putative LysR-type regulator
sequences were sourced from the website
http://bioinfo.genopole-toulouse.prd.fr/
annotation/iANT/bacteria/rhime/. ClustalW (http://www.
ebi.ac.uk/Tools/clustalw2/index.html) was used to align multiple
sequences and construct the evolutionary tree, and all default
parameters were selected.
Results
Six LysR family regulators belong to an LrhA sub- family The
deduced amino acid sequences of 94 LysR family regulators from S.
meliloti Rm1021 were sourced from the website
http://bioinfo.genopole-toulouse.prd.fr/anno-
tation/iANT/bacteria/rhime/. Those sequences were input into the
EBI ClustalW server to construct a phylogenetic tree. Six members
of the LysR family, that is, products encoded by mutants SMa1979,
SMb20715, SMc00820, SMc04163, SMc03975, and SMc04315, were located
on the same clade of the evolutionary tree (data not shown). Each
of these regulators showed approximately 30% ho- mology with E.
coli LrhA, Erw. carotovora HexA, or Pseu- domonas putida DgdR by
BlastP analysis (http://www.
ncbi.nlm.nih.gov/blast/Blast.cgi?PAGE=Proteins&
PROGRAM=blastp&BLAST_PROGRAMS=blastp&PAGE_
TYPE=BlastSearch&SHOW_DEFAULTS=on). Results of multiple
sequence alignments indicated that these six regu- lators were
highly homologous to LrhA, HexA, and DgdR, especially in the
DNA-binding domains, although the C- terminal domains were quite
variable in length (Fig. 1). These analyses revealed that these
genes could be classi- fied into an LrhA subfamily of the LysR
family of regula- tor genes.
Effect of the mutations of LrhA subfamily regulators on motility
The motility of six LysR-type regulator mutants was determined on
the LB/MC or ZMGS swimming agar plates. All mutants migrated more
slowly than the wild-type strain (Rm1021) on LB/MC agar (Fig. 2).
After 4 d of culture, the diameters of their colonies were
86.6%±0.5% to 93.5%±0.5% of that of Rm1021. On the ZMGS agar
plates, Sm341 had a slightly higher mobility than Rm1021 (data not
shown), whereas the other five mutants swam more slowly than the
wild type. This result indicated that mutation of these five genes
impairs their swimming mobility.
Effect of the mutations of LrhA subfamily regulators on AHL
accumulation The strain A. tumefaciens NTL4 (pZLR4) was used as an
indicator to measure the transcriptional level of traG (as
traG-lacZ fusion is controlled by AHL-like signals) [15], to assess
the relative amount of AHL in rhizobia culture. NTL4 (pAtC58) and
NTL4 (pAtC58, pTiC58accR) were used as the negative and positive
controls, respectively. The mutants Sm379, Sm382, and Sm383 showed
similar AHL concentrations to that with the wild type, although
Sm382 had a 4-fold increase at OD600 nm=1.45. Much lower levels of
AHL were found in the cultures of Sm326, Sm341, and Sm360 (Fig. 3).
These results suggested that Sm326, Sm341, and Sm360 were defective
in AHL production even in complete medium.
Effect of the mutations of LrhA subfamily regulators on
extracellular protein production Quantitative spectrophotometric
assays were carried out to assess the total extracellular proteins
produced by the mutants of LrhA subfamily regulator at three growth
phases. The level of total extracellular proteins produced by
SMa1979 mutant strain Sm326 became 1-fold, 4-fold, and 2-fold
higher than Rm1021 at OD600 nm=0.2, 2.0, and 5.0, respectively, but
the results on other mutants showed no such significant difference
(Fig. 4). The extracellular protein secretion was observed at much
higher levels in SMa1979 mutant strain Sm326 compared with a wild-
type strain control at an early stationary phase. These results are
very similar to those of hexA mutation in Erw. carotovora ssp.
carotovora [6].
SMa1979 mutation (Sm326) resulted in impairment in motility,
defects in AHL production, and increased secretion of extracellular
protein, as in the Erw. carotovora hexA mutant; and SMb20715,
SMc00820, and SMc03975 had
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Sinorhizobium meliloti lrhA subfamily genes
Fig. 1 Multiple sequence alignment of LrhA homologs in
Sinorhizobium meliloti Amino acid residues identical throughout the
LrhA subfamily are shaded in black, similar residues are shaded in
gray. Gaps are marked with “–” and subfamily names are indicated at
the beginning of each line. LrhA was from Escherichia coli, HexA
was from Erwinia carotovora, and DgdR was from Pseudomonas
putida.
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Sinorhizobium meliloti lrhA subfamily genes
Fig. 2 Motility of LrhA homologous gene mutants from Sinorhizobium
meliloti (A) A sample of 3 µl resuspended culture of wild-type
Rm1021 and mutants of the six LrhA homologs was spotted onto LB/MC
(Luria-Bertani medium supplemented with 2.5 mM MgSO4 and 2.5 mM
CaCl2) soft agar media (0.35%) and incubated at 30 ºC for 4 d. (B)
Relative diameters of each mutant colony compared with that of the
wild type.
Fig. 3 Level of N-acyl homoserine lactone (AHL) signal in
Sinorhizobium meliloti LrhA homolog mutants The levels of AHL
signal in cultures of different s trains were measured at OD600
nm=0.36, 1.45, and 2.00. NTL4 (pAtC58) and NTL4 (pAtC58,
pTiC58accR) were applied as the negative and positive controls,
respectively.
similar functions to E. coli lrhA.
LrhA subfamily genes in other bacteria As many bacterial genomes
have been sequenced and published, it is possible to search for
more LrhA subfamily genes and conveniently analyze their origin and
evolution.
The deduced amino acid sequence of E. coli LrhA was input into the
National Center for Biotechnology Information’s Blast/genome server
(http://www.ncbi.nlm. nih.gov/sutils/genom_table.cgi) to search for
homologous genes in other bacterial genomes (e<0.0001,
Score>80,
Fig. 4 Extracellular protein produced by Sinorhizobium meliloti
LrhA mutants Total extracellular protein produced by the wild type
(Rm1021) and mutants of the LrhA subfamily regulator were measured
at OD600 nm=0.2, 2.0, and 5.0.
Dec, 2004). The LrhA subfamily homologous genes were found in
bacteria, not in archaea. Furthermore, 140 genes were found in
proteobacteria, but only six genes in actinobacteria. Among the 140
genes found in proteobacteria, 39 genes were distributed in the α
subgroup, 65 genes in the β subgroup, and 36 genes in the γ
subgroup (Fig. 5), but none were found in either the δ or the ε
subgroups. In the α subgroup, there are LrhA homologs in Rhizobium,
such as the 6 genes in S. meliloti Rm1021, 10 genes in
Mezorhizobium loti MAFF303099, and 7 genes in A. tumefaciens C58
[Fig. 5(A)]. A large number of LrhA genes were found in
Burkholderia, for example, 17 genes in
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Sinorhizobium meliloti lrhA subfamily genes
Burkholderia cepacia R18194 [Fig. 5(B)]. However, there was only
one homolog found in most species of the γ subgroup; only
Pseudomonas aeruginosa PAO1 contained five members [Fig. 5(C)].
These results suggest that the distribution of the LrhA subfamily
is significantly different in different bacterial families.
Discussion
The known number of LysR family genes has increased
with the publication of bacterial genome sequences. Many
bacteriologists are interested in the functions of these regu-
lators in nature. Schell [1] wrote a review in 1993, but a lot of
new genes have since been found to have novel roles in metabolism,
symbiosis, and bacterial swimming, such as LrhA, HexA, and PecT
[4].
With an increase in the number of genome sequences, sequence
analysis is a preferred tool for analyzing func- tions of target
genes. A new subfamily belonging to the LysR family was suggested
because the amino acid se-
Fig. 5 Distribution of LrhA subfamily genes in proteobacteria LrhA
homologs in other bacterial genomes, sourced from the National
Center for Biotechnology Information’s BLAST/genome server
(http://www.ncbi.nlm.nih.gov/sutils/genom_table.cgi) (e<0.0001,
Score>80, Dec 2004). (A) Number of LrhA homologs in the α
subgroup. (B) Number of LrhA homologs in the β subgroup. (C) Number
of LrhA homologs in the γ subgroup. At, Agrobacterium tumefaciens;
Av, Alcanivorax borkumensis SK2; Bb, Bordetella bronchiseptica;
Bc1, Burkholderia cenocepacia; Bc2, Burkholderia cepacia; Bf,
Burkholderia phymatum; Bj, Bradyrhizobium japonicum; Bm, Brucella
melitensis; Bma, Burkholderia mallei; Bpa, Bordetella
parapertussis; Bpe, Bordetella pertussis; Bps, Burkholderia
pseudomallei; Bs, Brucella suis; Cv, Chromobacterium violaceum; Ec,
Escherichia coli 101-1; Eco1, Escherichia coli 536; Eco2,
Escherichia coli B171; Eco3, Escherichia coli K12; Il, Idiomarina
loihiensis; Lp1, Legionella pneumophila str. Corby; Lp2, Legionella
pneumophila str. Lens; Lp3, Legionella pneumophila str. Paris; Ml,
Mesorhizobium loti; Msp, Mesorhizobium sp. BNC1; Na,
Novosphingobium aromaticivorans; Pa1, Psychrobacter arcticus 273-4;
Pa2, Pseudoalteromonas atlantica; Pf, Psychromonas ingrahamii 37;
Pl, Photorhabdus luminescens; Pp, Psychrobacter sp. PRwf-1; Psy,
Psychrobacter cryohalolentis K5; Psy2, Psychromonas sp. CNPT3; Re,
Ralstonia eutropha; Rm, Ralstonia metallidurans; Rp,
Rhodopseudomonas palustris; Rs, Rickettsia sibirica; Rsc, Ralstonia
solanacearum; Se1, Salmonella enterica ssp. enterica serovar
Choleraesuis str. SC-B67; Se2, Salmonella enterica ssp. enterica
serovar Newport str. SL254; Sm, Sinorhizobium meliloti; Sp,
Silicibacter pomeroyi; Spsp, Sphingomonas sp. SKA58; St, Salmonella
typhimurium; Vp, Vibrionales bacterium SWAT-3; Vv, Vibrio
vulnificus; Yp1, Yersinia pestis Antiqua; Yp2, Yersinia pestis
Pestoides F .
Acta Biochim Biophys Sin (2008) | Volume 40 | Issue 2 | Page
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Sinorhizobium meliloti lrhA subfamily genes
quences shared similar identities with those of E. coli LrhA and
Erw. carotovora HexA. The sequences were also lo- cated on one
clade of the evolutionary tree, providing many clues to identify
the functions of these genes. The results of genome sequence
analyses suggest that this subfamily is distributed in some
bacterial species, but not all.
The motility of all mutants of the S. meliloti LrhA subfamily,
except Sm341, was impaired on both com- plete and minimum media. It
is interesting to note that the motility of the mutant SMb20715 was
different on these two media. It swam slower than the wild-type on
the LB/ MC medium [7], but quicker on the ZMGS medium. One
assumption is that lower nutrient supply might promote the
chemotaxis of rhizobia.
Furthermore, three gene mutants had fewer AHL signals, whereas the
mutant SMc03975 had a 4-fold increase. The effects of these
mutations will be determined in further studies. Sm326, an SMa1979
mutant, had significantly more extracellular protein than the wild
type. It is appar- ent that this mutant had phenotypes similar to
those of E. coli lrhA and Erwinia hexA.
It is interesting to study how these genes can affect the motility
of rhizobia and the relationship between the pro- duction of AHL
and motility. It has been reported that, in many bacteria, swarming
motility is quorum-sensing con- trolled [19]. It was shown that
AHL-dependent synthesis of the biosurfactants is required for
swarming motility [20− 23], although AHL-deficient mutants of
Pseudomonas syringae pv. syringae B728a had high motility [24]. In
S. meliloti, swarming of the mutant 8530 strain could be dependent
on SinI- and/or ExpR-mediated quorum sens- ing [25]. It might be
hypothesized that these genes affect the motility of rhizobia by
affecting the production of AHL, but this needs to be proven in
future works. Why did the mutant of SMa1979, Sm326, produce more
extracellular protein? The characteristics of these unknown
extracellu- lar proteins, and promoters of regulatory genes and
target genes will be investigated in our laboratory.
Acknowledgements
We thank Dr. Stephen Farrand (Department of Microbiology,
University of Illinois at Urbana-Champaign) for providing A.
tumefaciens NTL4 (pZLR4), NTL4 (pAtC58), and NTL4 (pAtC58,
pTiC58accR). We thank Prof. Tianduo Wang (retired) for revising the
manuscript.
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