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Original Paper
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Acta Biochim Biophys
Sin 2008, 40: 166-173 |
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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 Sciences, 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 encoding 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 a, b, and g subdivisions
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 transcriptional 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 motility-related genes [3]. The LrhA protein is highly homologous 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 required for lytic reactions [4,5].
Overexpression of the Erw. carotovora hexA gene in the opportunistic
human pathogen Serratia also represses multiple virulence determinants
[5]. In hexA mutants of Erw. carotovora, expression of flagella
genes (fliA and fliC) is increased, thereby resulting 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 pheromone 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 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 phylogeny 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 mutants 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 regulators 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 mg/ml; ampicillin, 100 mg/ml; neomycin, 200 mg/ml; streptomycin, 500 mg/ml; tetracycline, 10 mg/ml; spectinomycin, 100 mg/ml; and gentamicin, 50 mg/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 incubated 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 Gonz�lez [14] with some modifications. Briefly, 150 ml supernatant of bacteria culture was mixed with 30 ml indicator strain Agrobacterium tumefaciens NTL4 (pZLR4) [9]. NTL4 (pAtC58) and NTL4 (pAtC58, pTiC58DaccR) were used as the negative and positive controls, respectively [10,15]. The relative amount of AHL of those bacteria was determined by measuring the b-galactosidase activity of the indicator strain after 3 h. The b-galactosidase assays were carried out as described by Miller [16]. Spectrophotometer 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 subfamily
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/annotation/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% homology 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& �PRO�GRAM�=blastp&BLAST_PRO�GRAMS=blastp&PAGE_TYPE=BlastSearch&SHOW_DEFAULTS=on). Results of multiple sequence alignments indicated that these six regulators 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 classified into an LrhA subfamily of the LysR family of regulator 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, pTiC58DaccR) 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 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, 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 a subgroup, 65 genes in the b subgroup, and 36 genes in the g subgroup (Fig. 5), but none were found in either the d or the e subgroups. In the a 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 Burkholderia cepacia R18194 [Fig. 5(B)]. However, there was only one homolog found in most species of the g 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 regulators 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 functions of target genes. A new subfamily belonging to the LysR family was suggested because the amino acid sequences shared similar identities with those of E. coli LrhA and Erw. carotovora HexA. The sequences were also located 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 complete 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 apparent 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 production of AHL and motility. It has been reported that, in many bacteria, swarming motility is quorum-sensing controlled [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 sensing [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 extracellular 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, pTiC58DaccR). We thank Prof. Tianduo Wang (retired) for revising the manuscript.
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