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The LsrB protein is required for Agrobacterium tumefaciens
interaction with host plants
Guirong Tang1, 2
, Qiong Li1, Shenghui Xing
1, Ningning Li
1, Zheng Tang
1,
Liangliang Yu1, Junhui Yan
1, Xuan Li
3, Li Luo
1
1Shanghai Key Laboratory of Bio-energy Crops, Center of Plant Science, School of
Life Sciences, Shanghai University, Shanghai 200444, China; 2School of
Communication & Information Engineering, Shanghai University, Shanghai 200444,
China; 3Key Laboratory of Synthetic Biology, Institute of Plant Physiology and
Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences,
Shanghai 200032, China
Running title: Regulation of A. tumefaciens lsrB upon infection
Author for correspondence:
Li Luo
Tel.:+86 21 66135321
Email: [email protected]
Key words: LsrB; Agrobacterium tumefaciens; reactive oxygen species; attachment;
exopolysaccharide; infection; gene expression
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Abstract
Agrobacterium tumefaciens infects and causes crown galls in dicot plants by
transferring T-DNA from the Ti plasmid to the host plant via a type IV secretion
system (T4SS). This process requires appropriate environmental conditions, certain
plant secretions and bacterial regulators. In our previous work, a member of the LysR
family of transcriptional regulators (LsrB) in Sinorhizobium meliloti was found to
modulate its symbiotic interactions with the host plant alfalfa. However, the function
of its homologue in A. tumefaciens remains unclear. In this study, we show that the
LsrB protein of A. tumefaciens is required for efficient transformation of host plants.
An lsrB deletion mutant of A. tumefaciens exhibits a number of defects, including in
succinoglycan production, attachment, and resistance to oxidative stress and iron
limitation. RNA-sequencing analysis indicated that 465 genes were significantly
differentially expressed (upregulation of 162 genes and downregulation of 303 genes)
in the mutant compared to the wild-type strain, including those involved in
succinoglycan production, iron transporter and detoxification enzymes for oxidative
stress. Moreover, expression of the lsrB gene from S. meliloti, Brucella abortus or A.
tumefaciens rescued the defects observed in the S. meliloti or A. tumefaciens lsrB
deletion mutant. Our findings suggest that a conserved mechanism of LsrB function
exists in symbiotic and pathogenic bacteria of the family Rhizobiaceae.
Introduction
Agrobacterium tumefaciens is a facultative plant pathogen that induces crown gall
diseases in dicot plants. During infection, a DNA fragment (T-DNA) from the A.
tumefaciens Ti plasmid is transferred to plant cells and integrates into the plant
genome; the transgene-encoded proteins drive the synthesis of phytohormones,
cytokinins and auxin, which elicit plant cell proliferation and tumourigenesis (White
and Braun, 1941; Thomashow et al., 1984). This process requires the VirB/D4 type IV
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secretion system (T4SS) of A. tumefaciens. Because of its ability to transfer DNA to
plants, A. tumefaciens has been developed as an important tool in plant genetic
transformation and engineering (Azpiroz-Leehan and Feldmann, 1997).
The virulence (vir) gene located on the Ti plasmid is essential for T-DNA transfer and
tumorigenesis. A. tumefaciens possesses a VirA/VirG two-component regulatory
system that senses important environmental signals, including acidity,
monosaccharides, and phenolic compounds such as acetosyringone (AS), which are
released by wounded plant cells, to regulate vir gene induction (Shimoda et al., 1990;
Palmer et al., 2004; Brencic and Winans, 2005; Bhattacharya et al., 2010; Pitzschke
and Hirt, 2010; Lacroix and Citovsky, 2013). Plants generate high levels of reactive
oxygen species (ROS), such as H2O2 and superoxide radicals, which are employed in
an important initial defence mechanism to inhibit bacterial invasion and proliferation
(Baker and Orlandi, 1995). Catalase (CAT) and superoxide dismutase (SOD) are
crucial antioxidant enzymes that degrade H2O2 and superoxides, respectively. These
enzymes have been shown to serve as virulence factors involved in A. tumefaciens
tumorigenesis (Xu and Pan, 2000; Saenkham et al., 2007). Iron ions are also key
players in maintaining redox homeostasis in bacteria. Homologous iron-sensing
regulatory ferric uptake regulation (fur) genes from plant pathogens have been shown
to play a critical role during plant-pathogen interaction (Franza et al., 1999;
Subramoni and Sonti, 2005). In addition, plant mechanisms for depriving invading
microbes of iron have been suggested (Neema et al., 1993). Therefore, plant
pathogens need to overcome both oxidative stress and iron deprivation for survival
and proliferation in host plants. The A. tumefaciens catalase KatA is an important
virulence factor involved in the detoxification of H2O2 released from plant tumours
(Xu and Pan, 2000; Xu et al., 2001). Expression of katA in A. tumefaciens is regulated
by OxyR, a LysR family transcriptional regulator that is responsible for bacterial
resistance to oxidative stress (Nakjarung et al., 2003). A. tumefaciens, B. abortus and
S. meliloti are members of Rhizobiaceae family and these are conserved in a much
larger family, such as OxyR/KatA homologues (Jamet et al., 2005).
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In our previous work, a new LysR family transcriptional regulator gene, lsrB, required
for nodulation and nitrogen fixation (Luo et al, 2005) was identified in Sinorhizobium
meliloti. A lsrB deletion mutant induced the formation of inefficient nitrogen-fixing
root nodules on host alfalfa plants (Medicago sativa) (Tang et al., 2013). As LsrB
directly regulates the expression of genes involved in lipopolysaccharide biosynthesis
and glutathione production (Lu et al., 2013; Tang et al., 2014), it constitutes a new
redox regulator in S. meliloti (Tang et al. 2017). A homologue of lsrB has also been
identified in Brucella species (Foulongne et al., 2000; Sheehan et al., 2015). However,
the function of LsrB in A. tumefaciens has not yet been determined.
In this work, we constructed an A. tumefaciens lsrB deletion mutant and examined it
for both free-living and pathogenic phenotypes. We identified new functions of LsrB
in A. tumefaciens and also compared the functions and mechanisms of three LsrB
proteins from B. abortus, S. meliloti and A. tumefaciens. A conserved mechanism of
LsrB in all three species is proposed.
Results
Expression of the pathogenic bacterial lsrB gene rescued the symbiotic defects of
the S. meliloti lsrB mutant.
Homologous lsrB genes have been identified in several bacterial species, including
members of Sinorhizobium and Brucella (Tang et al., 2013; Sheehan et al., 2015). The
LsrB proteins from A. tumefaciens and B. abortus share 88% and 69% identity with
the S. meliloti LsrB protein, respectively. These proteins are composed of a
DNA-binding domain and a LysR substrate-binding domain (Fig. 1A and Fig. S1),
and the observed protein sequence identity suggests that they may share the same
function or mechanism of action.
To assess this possibility, the S. meliloti lsrB deletion (∆lsrB) mutant, which induces
defective nitrogen-fixation nodules on alfalfa, was used in a symbiotic nodulation
assay with alfalfa plants (Tang et al., 2013). In Rhizobium-legume symbiosis, mutants
that induce less effective nitrogen -fixation nodules can induce the production of a
larger number of the plant organs (Oka-Kira and Kawaguchi, 2006). The lsrB gene
from A. tumefaciens or B. abortus was expressed in the S. meliloti ∆lsrB mutant, and
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each strain was inoculated onto alfalfa seedlings. After four weeks, the growth of
plants inoculated with the lsrB deletion mutant expressing the lsrB gene from either A.
tumefaciens or B. abortus was similar to that of plants inoculated with the wild-type
strain of S. meliloti (Rm1021) (Fig. 1B). Additionally, the biomass (dry weight) and
number of nodules of plants induced by the mutant expressing lsrBat or lsrBba were
found to be similar to those induced by Rm1021 (Fig. 1C-D). These results indicate
that the lsrB gene from A. tumefaciens or B. abortus completely rescued the symbiotic
defects of the S. meliloti lsrB deletion mutant, suggesting that these LsrB proteins
have conserved physiological functions or molecular mechanisms in both symbiotic
and pathogenic bacteria.
The A. tumefaciens ∆lsrB mutant is deficient in host plant transformation.
To determine new functions of the lsrB gene during interactions with hosts, an A.
tumefaciens lsrB deletion mutant was constructed using a suicide-plasmid
homologous recombination method (see Materials and Methods). For the
complementation analysis, a plasmid expressing the lsrB gene from A. tumefaciens, B.
abortus or S. meliloti was introduced into the ∆lsrB mutant. In addition, a
plant-expression vector pCAMBIA2301 (Cambia, Canberra, Australia) carrying a
CAMV-35S driven-GUS gene was transferred into each A. tumefaciens strain for the
plant transformation assay. Roots of young Arabidopsis seedlings were treated with
each strain, and β-glucuronidase (GUS) activity in wounds was determined. The
results showed that the lsrB deletion mutant induced fewer (less than 1/10 as many)
transformation events than the parent strain C58 C1, whereas the plasmid expressing
the lsrB gene from A. tumefaciens, B. abortus or S. meliloti completely rescued the
defect of the mutant (Fig. 2A-F). These results suggest that LsrB is required for A.
tumefaciens transformation of host plants.
The lsrB gene is required for efficient attachment of A. tumefaciens onto host
plants.
As bacterial attachment is a key step in the infection of a host plant by A. tumefaciens
(Lippincott and Lippincott, 1969), we therefore evaluated the attachment efficiency of
the A. tumefaciens lsrB deletion mutant onto Arabidopsis roots. Roots were soaked in
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fresh cell suspensions of A. tumefaciens constitutively expressing green fluorescent
protein (GFP), and attached cells were analysed under a fluorescence microscope after
two hours. Compared to Arabidopsis roots inoculated with the parent strain C58C1,
weaker fluorescence intensity was observed in the Arabidopsis roots inoculated with
the lsrB deletion mutant (Fig. 3A-B). This defect was rescued by expressing the lsrB
gene from A. tumefaciens, B. abortus or S. meliloti (Fig. 3C-E). The numbers of
bacterial cells attached to the plant roots were counted by diluting and plating samples;
significantly fewer cells of the lsrB deletion strain were present compared to the
parent strain C58C1, and higher counts were observed after introducing the lsrB gene
from any of the three bacterial species (Fig. 3F). These results suggest that LsrB is a
positive regulator of Agrobacterium attachment onto host plants.
The LsrB protein positively regulates succinoglycan and biofilm production in A.
tumefaciens.
The A. tumefaciens lsrB deletion mutant grew slightly slower in liquid medium
compared with the parent strain C58C1, and this defect was rescued by the expression
of the lsrB gene from A. tumefaciens, B. abortus or S. meliloti (Fig. S2). Interestingly,
the colonies of the lsrB deletion mutant did not appear mucoid, which prompted us to
evaluate exopolysaccharide (EPS, succinoglycan) production by the mutant. The
fluorescent stain Calcofluor White M2R, which can specifically bind to succinoglycan,
was used to evaluate succinoglycan production by A. tumefaciens, showing that the
colonies of the lsrB deletion mutant produced weaker fluorescence than did the
wild-type strain (Fig. 4A). The introduction of plasmids expressing the lsrB genes
from A. tumefaciens, B. abortus or S. meliloti restored succinoglycan production in
the lsrB deletion mutant (Fig. 4A). Quantitative analysis of the fluorescence intensity
revealed that 5-fold more succinoglycan was produced by the parent and
complemented strain compared to the lsrB deletion mutant (Fig. 4B). The total sugar
content in the exopolysaccharide produced by A. tumefaciens growing in broth was
analysed using an anthrone sulphuric acid method, and the results revealed
significantly decreased total sugar for the lsrB mutant compared with the parent or
complemented strain (Fig. 4C). Additionally, biofilm formation by each A.
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tumefaciens strain in tubes was examined. Based on visual inspection, the lsrB mutant
formed only a small amount of biofilm, whereas the parent and complemented strains
produced a large amount of biofilm. Furthermore, quantification of the biofilm using
crystal violet showed less biofilm on tubes formed by the lsrB deletion mutant
compared to the parent or complemented strain (Fig. 4D). These results indicate that
the LsrB protein positively regulates succinoglycan production and biofilm formation
in A. tumefaciens.
The LsrB protein is essential for A. tumefaciens adaptation to oxidative stress
and iron limitation.
The increased expression of KatA exhibited by the S. meliloti lsrB deletion mutant
(Tang et al., 2013) suggests that this mutant is sensitive to oxidative stress. Similarly,
in the present study, we hypothesized that the A. tumefaciens lsrB deletion mutant
may also be sensitive to these oxidizing agents. To test this possibility, cell sensitivity
assays using hydrogen peroxide (H2O2) and sodium nitroprusside (SNP) were
performed. A. tumefaciens ∆lsrB cells were found to be hypersensitive to 10 mM
H2O2 and 10 mM SNP (Fig. 5), and this defect was rescued by expressing of the lsrB
gene from A. tumefaciens, B. abortus or S. meliloti (Fig. 5). These results suggest that
the ability of A. tumefaciens to resist oxidative stress is positively influenced by the
LsrB protein.
Bacterial adaptation to oxidative stress is associated with intracellular redox
homeostasis, which is influenced by levels of iron ions. To determine whether A.
tumefaciens lsrB contributes to iron transport, sensitivity to Dipy (2, 2-dipyridyl, a
metal ion chelator) was analysed. Our data indicated that the lsrB deletion mutant was
hypersensitive to Dipy both on solid and liquid media, and this defect was rescued by
expressing the lsrB gene from A. tumefaciens, S. meliloti or B. abortus (Fig. 6).
Moreover, addition of FeCl3 was able to restore the resistance of the mutant to Dipy
(Fig. 6). These data indicate that LsrB is a positive regulator of iron homeostasis in A.
tumefaciens.
The lsrB deletion mutant exhibits decreased survival in infiltrated tobacco
leaves.
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To confirm that LsrB is required for A. tumefaciens transformation of host plants,
young tobacco leaves were used for infiltration experiments with suspensions of
bacteria harbouring the GUS reporter gene. Staining results indicated a lower level of
GUS expression in leaves infiltrated with the lsrB deletion strain than in leaves
infiltrated with the parent strain C58 C1 at the same bacterial density (Fig. 7A). It is
well known that plant wounds release several chemicals, including ROS (superoxide
anions and hydrogen peroxide) (Baker and Orlandi, 1995). To determine factors
affecting transformation efficiency, nitrobluetetrazolium (NBT) staining was used to
assess superoxide anion radical accumulation. The data confirmed that infiltrated
tobacco leaves produced large amounts of O2- (Fig. 7B-C). In addition, the bacteria
infiltrated into tobacco leaves were recovered, plated and counted. Starting one day
after inoculation, we detected a lower recovered bacterial cell density of the lsrB
deletion mutant than with the parent strain (Fig. 7D and Fig. S3), suggesting that the
proliferation or survival of A. tumefaciens cells in the wounds of host plants requires
the LsrB protein and may be associated with ROS sensitivity.
Downstream gene expression in the A. tumefaciens lsrB deletion mutant.
Due to the multiple phenotypic defects induced by lsrB deletion in A. tumefaciens, it
seemed plausible that multiple genes are differentially expressed in the mutant
compared with the parent strain. To assess this possibility, the whole-genome
transcriptomes of both the deletion mutant and the parent strain were analysed by
RNA-sequencing (RNA-Seq) (Table S3), and genes identified as being differentially
expressed were selected for confirmation by quantitative reverse transcription
polymerase chain reaction (RT-qPCR). The transcript levels of 14 of the selected
genes (attD, exoV, exoC, exoP, oxyR, trxA, Atu3676, Atu3679, Atu3680, Atu3688
(fecB), Atu3690 (fecD) and Atu3691 (fecE)) were decreased in the lsrB deletion
mutant, and oxidative stress (katA) and succinoglycan production (exoR) were
increased in the lsrB deletion mutant (Fig. 8). These data indicate that the expression
of several genes associated with adaptation to oxidative stress (oxyR, trxA),
succinoglycan production (exoV, exoC, exoP), siderophore biosynthesis (fecB, fecD,
fecE) and iron transporter (Atu3676, Atu3679, Atu3680) is positively regulated by the
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LsrB protein.
Discussion
A. tumefaciens causes crown gall on plants and is used in plant genetic engineering.
Transformation of plant cells via A. tumefaciens depends on bacterial adaptation to
the environment of plant wounds. Indeed, bacteria have developed several strategies
to perceive environmental signals from plant wounds to reprogramme their gene
expression. Members of the LysR-type family of regulators are able to sense
environmental cues and function as key players in these processes (Schell, 1993;
Maddocks and Oyston, 2008). The lsrB gene, which was first identified in B. suis
(Foulongne et al., 2000) and later in S. meliloti (Luo et al., 2005), is essential for
alfalfa nodulation and nitrogen fixation by S. meliloti (Tang et al., 2013) and for
Brucella infection and survival in a human cell line and mice (Tang et al., 2013;
Sheehan et al., 2015). In this study, we found that LsrB, an A. tumefaciens LysR
family regulator, overcomes both oxidative stress and iron deprivation and controls
transformation efficiency in host plants. These findings are important for
understanding the regulatory mechanism of the interaction of A. tumefaciens with its
hosts.
For many years, att genes were proposed to mediate attachment and were also
reported to be required for virulence (Matthysse et al., 2000). However, the genome
sequence of A. tumefaciens C58 (Goodner et al., 2001; Wood et al., 2001) revealed the
att cluster to be located on the plasmid pAtC58, which is known to be dispensable for
virulence (Hynes et al., 1985). Analysis using isogenic derivatives revealed that
pAtC58 has only mild effects on virulence gene expression, with no obvious impact
on attachment (Nair et al., 2003). Although our data do not address whether the att
genes are directly associated with attachment, we note that it did find significantly
lower expression of attD as well as diminished attachment with the lsrB mutant (Figs.
3 and 8). Additionally, the avhB genes, these encode the T4SS that mediates
conjugation of pAtC58 was also decreased in the lsrB mutant (Table S3), this could
possibly be relevant to the diminished attachment.
Based on the results of our study, LsrB is a positive regulator of exopolysacchride
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(including succinoglycan) production (Fig. 4), and we confirmed that LsrB positively
regulates succinoglycan biosynthesis by activating exo gene expression (Fig. 8). For
example, expression of exoR was upregulated in the lsrB deletion mutant. ExoR acts
as a negative regulator of succinoglycan biosynthesis by suppressing the ChvG/ChvI
two-component system, which directly activates expression of exo genes in both S.
meliloti and A. tumefaciens (Wells et al., 2007; Chen et al., 2008; Wu et al., 2012).
Therefore, our data suggest that increased levels of ExoR led to the repression of the
exo genes via ChvG/ChvI.
The plant transformation deficiency of the lsrB deletion mutant might largely result
from the decrease in bacterial adaptation to the environment. In this study, we found
the free-living lsrB mutant to be sensitive to oxidants (Fig. 5), which is consistent
with the observations in plants (Fig.7). Upon wounding, an oxidative burst is induced
in plants (Fig. 7B-C), and the sensitivity of the mutant to this condition may lead to
bacterial cell death (Fig. 7D). We also observed the mutant to be hypersensitive to
iron limitation (Fig. 6). Iron transport plays a crucial role in redox homeostasis (Imlay
et al., 1988), and iron limitation has been suggested to occur during bacterium-plant
interactions (Neema et al., 1993; Mila et al., 1996); thus, iron-limitation sensitivity
may also contribute to bacteria survival in wounds. Consistently, the expression of
genes related to the oxidative response and detoxification and iron transporters was
correspondingly altered in the mutant (Fig. 8).
The regulatory mechanism of LsrB in bacteria may be conserved. First, the lsrB gene
from B. abortus, A. tumefaciens or S. meliloti can complement the lsrB deletion
mutant phenotype in A. tumefaciens and S. meliloti (Figs. 1-6). Second, the LsrB
protein could directly regulate expression of lrp3 (though function of this gene is not
clear) in both S. meliloti and A. tumefaciens, as conserved TN11A boxes are predicted
to be present in the lrp3 promoter (Fig. S4) and the LsrB proteins directly binds to the
lrp3 promoter region in S. meliloti (Tang et al., 2014). In summary, our results reveal
that LsrB is a global regulator in the interaction of A. tumefaciens with host plants, a
finding that is important for understanding the molecular mechanism and evolution of
bacterial adaptation to biotic environments.
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Materials and Methods
Bacterial strains, plasmids, and growth conditions.
All bacterial strains and plasmids used in this study are listed in Table S1. The
reagents and medium components were obtained from Dingguo Corporation (Beijing,
China) and Sigma-Aldrich (Mainland, China). All restriction enzymes and molecular
biology reagents were purchased from TakaRa (Dalian, China). DNA sequences were
determined by Shanghai RuiDi Biological Technology Corporation. The
oligonucleotide primers used in this study are listed in Table S2. Escherichia coli was
grown aerobically at 37°C in Luria-Bertani (LB) medium (Sambrook et al., 1989). A.
tumefaciens were grown aerobically at 28°C in yeast tryptone (TY) medium (Beringer,
1974) or Luria-Bertani (LB) medium. Antibiotics were used at the following
concentrations: chloramphenicol, 10 µg mL-1
; kanamycin, 50 µg mL-1
; gentamicin, 10
µg mL-1
; streptomycin, 500 µg mL-1
; rifampicin, 50 µg mL-1
and timentin, 200 µg
mL-1
.
Construction of deletion strains.
The constructed plasmids and primers used in this study are listed in Tables S1 and S2,
respectively. Fragments upstream and downstream of lsrBat were amplified using
primers P1 and P2 (upstream) and P3 and P4 (downstream). Primer P2 was designed
such that the 5’ sequence had reverse complementarity to primer P3. The
complementary sequence of these two primers facilitated splicing by overlapping the
extension of the two PCR products. Both flanking sequences were amplified and gel
purified. The two purified products were then used as both templates and primers (P1
and P4) in a second PCR reaction to generate the final spliced product, which was
ligated to pmD18 (TakaRa) and sequenced. The deletion construct was then excised
using restriction enzymes and ligated to the suicide vector pk18mobsacB (Schafer et
al., 1994) to produce plasmid pA1. The pk18mobsacB plasmid confers kanamycin
resistance and sucrose sensitivity. Plasmid pA1 was introduced into A. tumefaciens
C58C1 (Jelenic et al., 2000) via mating using MT616 (Finan et al., 1984), and A.
tumefaciens C58C1 was obtained from Shanghai Weidi Biotechnology Co., Ltd.
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Exconjugants with kanamycin resistance were screened on LB plates and selectively
grown on fresh LB plates containing 13% sucrose. The resulting colonies were tested
for neomycin sensitivity. Corrected lsrBat mutants were confirmed by PCR using
primers (P5/P6) flanking the deletion site.
Plasmid expression constructs.
To construct a plasmid for complementation of the ∆lsrBat mutant, an lsrB DNA
fragment that included the native promoter was PCR amplified from the genomic
DNA of A. tumefaciens C58C1 using primers P9/P10. The PCR product was cloned
into pSRK (Khan et al., 2008), and the plasmid was named plsrBat. The B. abortus
2308 lsrB homologue, including its native promoter, was amplified using primers
P7/P8 and cloned into pSRK; the plasmid was named plsrBba. The S. meliloti 1021
lsrB homologue, including its native promoter, was amplified using primers P11/P12
and cloned into pSRK; the plasmid was named plsrBsm. The constructs were
introduced into the ∆lsrBat strain via conjugation.
RNA extraction.
Subcultures were started by inoculation of 50 mL of fresh TY medium with 5% of
overnight cultures and allowed to grow to log phase (OD600 = 0.5), RNA was isolated
using TRIZOL Reagent (Invitrogen, 15596026) and cDNA was prepared using a kit
(TakaRa, 0047A). An Agilent 2100 Bioanalyzer was used to assess the integrity of the
RNA samples. RNA-Seq was carried out by Shanghai Bohao Biological Technology
Corporation. The accession number for the RNA-seq date is GEO: GSE108845.
qPCR primers were designed to amplify approximately 200-bp sequences from the
indicated genes. The primer sequences are provided in Table S2. The qPCR procedure
was performed with SYBR Green reagent (YEASEN, Shanghai, China). Sample
values were normalized using a ropD primer set and calibrated against the results for
wild-type.
Nodulation tests.
Nodulation assays using alfalfa plants were performed as reported previously (Tang et
al., 2013). Briefly, alfalfa seeds were surface sterilized and germinated. Alfalfa
seedlings were soaked for 30 min in a diluted bacterial solution (OD600 = 0.05), and
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transferred to pots filled with a mixture of autoclaved vermiculite and perlite (3:1).
After inoculation, the number of nodulated plants and the dry weight of shoots were
recorded for four weeks. The plants were watered with autoclaved nitrogen-free liquid
medium every 3 days.
Transient GUS activity assays of Arabidopsis plants.
Transient Agrobacterium-mediated transformation assays were performed as
described by Nam et al. (Nam et al., 1999) and Mysore et al. (Mysore et al., 2000).
For transient GUS activity assays (Mestiri et al., 2014), root segments from
10-day-old seedlings grown in vitro were pooled and co-cultivated with A.
tumefaciens harbouring the binary vector pCAMBIA2301 (Cambia, Canberra,
Australia) in hormone-free Murashige and Skoog (MS) medium for 48 h at 22°C.
Next, the seedlings were washed once in hormone-free MS medium and then cultured
in hormone-free MS medium supplemented with timentin for an additional 4 days to
kill the bacteria. Roots were subsequently stained with GUS buffer (100 mM
Na2HPO4, 100 mM KH2PO4, 10 mM EDTA, 0.5 mM K3Fe(CN)6, 0.5 mM K4Fe(CN)6,
1 mg mL-1
x-gluc, pH 7.0) overnight at 37°C. The stained samples were rinsed three
times with 1× phosphate-buffered saline (PBS) and cleared in different concentrations
of ethanol. The number of blue spots per root segment was counted, and three
independent experiments were performed.
Plant attachment assays.
Arabidopsis root attachment assays were carried out as previously described
(Tomlinson et al., 2010), with minor modifications. Roots of 5-day-old A. thaliana
seedlings grown on MS agar medium were transferred to sterile dishes containing 15
mL 1 mM CaCl2 and 0.4% sucrose. Next, the seedlings were inoculated with OD600 =
0.01 of the appropriate derivative strain of A. tumefaciens carrying the GFP reporter
plasmid pHC60 (Cheng and Walker, 1998). Ten root segments were inoculated per
strain. After 2 h of incubation in the dark at room temperature, the root segments were
rinsed and resuspended in fresh calcium chloride/sucrose solution. Five roots were
used to count the number of bacteria (colony-forming units (CFU) mL-1
), and another
five roots were sealed under coverslips. Fluorescence microscopy was performed
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under a Nikon microscope.
Transient GUS activity assays of tobacco plants.
A. tumefaciens cells harbouring the binary vector pCAMBIA2301 were harvested by
centrifugation. The cells were suspended in sterile water to an optical density OD600 of
0.2, corresponding to approximately 2 × 107
colony-forming units (CFU) per mL.
Half of a leaf (four weeks) was infiltrated with the A. tumefaciens C58C1 bacterial
suspension through the stomata on the abaxial side using a 1 mL plastic syringe
(without a needle). The second half of the leaf was inoculated with the ∆lsrBat
mutant. After 2 days, the leaves were stained with with x-gluc and
Nitrobluetetrazolium (NBT ) buffer.
Surviving bacterial counts.
To determine bacterial growth in tobacco leaves, wild-type tobacco plants (six weeks
old) were syringe inoculated with the C58C1 or ∆lsrBat strain (5 × 106 CFU/mL). The
internal bacterial number was determined at several time points (after 0, 1, 2, 3, 4 dpi)
via surface sterilization of a 1-cm2 section of inoculated plants with 5% H2O2 for 3
min (Ishiga and Ichinose, 2016). After washing three times with 100 µL sterile
distilled water, the plants were homogenized in sterile distilled water and the diluted
samples were plated on LB medium. Two to three days after plating of the diluted
samples, the bacterial CFU were counted. Bacterial numbers were evaluated in three
independent experiments.
Assay of superoxide anion radicals.
NBT (Sigma) was used to detect superoxide. Samples were stained with 3.5 mg/mL
NBT in 0.1 M potassium phosphate at pH 7.0 and vacuum-infiltrated three times (10
min each) in NBT staining solution. After infiltration, leaves were stained for 2 h at
room temperature followed by a wash with distilled water. The samples were then
cleared in 75% ethanol.
Assay for succinoglycan or EPS levels.
A. tumefaciens colonies were inoculated into LB liquid medium and grown overnight
at 28°C with shaking. The cultures were normalized to an OD600 of 0.2, and 5 µL was
spotted onto LB plates supplemented with 0.02% Calcofluor White M2R (Sigma).
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The plates were incubated for 2 days at 28°C, and succinoglycan production was
visualized under visible and UV light (Yao et al., 2004; Wang et al., 2013). All strains
were imaged using the same exposure value. The fluorescence intensity was analysed
using ImageJ Software. Next, 0.25 mL of A. tumefaciens strains, cultured to stationary
phase in LB, and 0.75 mL of isopropanol was added to precipitate EPS. The pellet
was washed once with 70% ethanol, air dried, redissolved in 0.25 mL of distilled
water, and used for total EPS measurements using the sulphate-anthrone method
(Wang et al., 2010).
Biofilm formation.
Biofilm formation in glass tubes was investigated using the method described by
Calatrava-Morales et al. (Calatrava-Morales et al., 2017), with modification. Briefly,
outgrown cultures of the tested strains were diluted 100-fold in glass tubes containing
4 mL fresh LB broth. The tubes were incubated for 48 h at 28°C under shaking
conditions for quantification of biofilm levels. After incubation, liquid from the tubes
was removed, and the bacterial biofilms were stained with 4 mL 0.1% crystal violet
solution (Sigma-Aldrich) for 20 min. The tubes were carefully washed three times
with deionized water to eliminate excess crystal violet. The crystal violet was
solubilized by the addition of 2 mL acetic acid (30% v/v), and absorbance at 590 nm
was used for quantification of the solubilized crystal violet stain.
Assay of oxidative stress sensitivity.
The sensitivity of A. tumefaciens to SNP was examined by growing cells in LB
medium at 28°C with shaking at 200 rpm/min to OD600 = 0.5, followed by
centrifugation. The cultures were serially diluted tenfold in LB broth, and aliquots (5
µL) were spotted onto LB agar supplemented with 10 mM H2O2 or 10 mM SNP to
determine the oxidative resistance of the strains at 28°C for 4 d.
Sensitivity to Dipy.
Overnight cultures grown in TY medium were washed once with fresh TY medium;
the cells were diluted to an OD600 of 0.05, and an iron-limiting condition was
achieved by adding 0.1 mM concentration of the iron chelator 2,2-dipyridyl (Dipy;
Sigma). Growth was monitored by measuring the OD600 after incubation at 28°C with
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shaking for 36 h. The effect of addition of metal ions on bacterial growth in the
presence of Dipy was assayed on TY agar plates. Overnight cultures grown in TY
medium were washed and adjusted to an OD600 of 0.03 in TY medium. Tenfold serial
dilutions were made. An aliquot (5 µL) of each dilution was spotted onto TY agar
plates containing 0.2 mM Dipy and 0.1 mM of FeCl3 and then incubated at 28°C for 4
d. Cells spotted onto an TY agar plate were used as a control.
Growth curves.
Bacteria were cultured in LB broth, and cells in the exponential growth phase were
diluted in fresh medium to OD600 = 0.05. The cell density (optical density at 600 nm
[OD600]) was monitored at each time point.
DNA sequence analysis.
The deduced protein sequence of LsrB was downloaded from the Sinorhizobium
meliloti genome site (http: //iant.Toulouse.inra.fr/bacteria/annotation/cgi/rhime.cgi).
Homologues of LsrB were aligned and downloaded from NCBI Blast Microbial
Genomes (http: //www.ncb i.nlm.nih.gov/sutils/genome_table.cgi).
Acknowledgements
This research was supported by the Natural Science Foundation of China (31570241
to L. L., 31500058 to G. T. and 31500197 to L. Y.) and the Shanghai Key Program of
Supporting (15230500100 to L. L.). We thank Dr. Shengqing Yu for gift of B. abortus
2308 genomic DNA.
Author contributions
L. L. and G. T. designed research; G. T., S. X., Q. L., N. L., L. Y., J. Y., and T. Z.
performed research; X. L., G. T., and L. L. analyzed data; L. L. and G. T. wrote the
paper.
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Legends
Fig. 1. Conservation among LsrB proteins from S. meliloti, A. tumefaciens and B.
abortus. (A) Domain organization of LsrB proteins from S. meliloti, A. tumefaciens
and B. abortus. LsrB proteins consist of DNA-binding and substrate-binding domains.
LsrB homologues from A. tumefaciens and B. abortus share 88% and 69%,
respectively, similarity in amino acid sequence with the protein from S. meliloti,
respectively. (B) An alfalfa plant growth after inoculation of the S. meliloti ∆lsrB
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strain expressing an A. tumefaciens or B. abortus. lsrB homologue. (C) Nodule
numbers of alfalfa plants inoculated with S. meliloti strains. (D) Biomass of alfalfa
plants inoculated with S. meliloti strains. Experiments were performed three times.
Forty-five alfalfa seedlings were inoculated with each S. meliloti strain. Vertical bars
indicate the standard error for three independent experiments. An asterisk denotes a
significant difference from Rm1021/vec in a t-test (* < 0.05 ). ∆lsrB, the deletion
mutant of lsrB in the S. meliloti 1021 background; plsrBat, the lsrB gene of A.
tumefaciens C58C1 expressed from the plasmid pSRK-Gm; plsrBba, the lsrB gene of
B. abortus expressed from the plasmid pSRK-Gm; vec, the plasmid pSRK-Gm.
Fig. 2. Transformation of Arabidopsis of wounded roots by A. tumefaciens strains.
(A-E) Arabidopsis root segments transformed by different A. tumefaciens strains
carrying pCAMBIA2301. (F) Mean numbers of blue spots in Arabidopsis root
segments induced by each different A. tumefaciens strain. Experiments were
performed three times. Over 50 Arabidopsis seedlings were inoculated with each A.
tumefaciens strain carrying pCAMBIA2301. Vertical bars indicate the standard error
for three independent experiments. An asterisk denotes a significant difference from
C58C1/vec in a t-test (* < 0.05 ). Bars, 1 mm.
Fig. 3. Attachment to Arabidopsis roots by A. tumefaciens strains. (A-E)
Attachment of A. tumefaciens cells to Arabidopsis roots. (F) Number of bacteria
attached to Arabidopsis roots. Experiments were performed three times. Over 20
Arabidopsis seedlings were inoculated with each A. tumefaciens strain carrying
pHC60 (a constitutive GFP construct). Vertical bars indicate the standard error for
three independent experiments. An asterisk denotes a significant difference from
C58C1/vec in a t-test (* < 0.05 ). Bars, 1 mm.
Fig. 4. Exopolysaccharide (EPS) production and biofilm formation by A.
tumefaciens strains. (A) EPS stained by Calcofluor White. OL, Bacterial colonies
under optical light; UV, bacterial colonies under UV. (B) Fluorescence intensity of
EPS was analysed using ImageJ software. (C) Quantification of the total EPS
produced by A. tumefaciens strains in LB broth. The sulphate-anthrone method was
used. This experiment was performed three times. Vertical bars indicate the standard
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error for three independent experiments. An asterisk denotes a significant difference
from C58 C1/vec in a t-test (* < 0.05 ) (D). Biofilm produced by bacterial strains
using a crystal violet staining method. Vertical bars indicate the standard error for
three independent experiments. An asterisk denotes a significant difference from
C58C1/vec in a t-test (* < 0.05 ).
Fig. 5. Sensitivity of A. tumefaciens strain to oxidants. A. tumefaciens strains were
cultured to OD600 = 0.5, diluted and spot inoculated onto LB medium containing either
10 mM H2O2 or 10 mM SNP. The experiment was performed three times.
Fig. 6. Sensitivity of the A. tumefaciens strain to Dipy. (A) Effect of the addition of
metal ions to growth in the presence of Dipy. Cells were diluted and spotted onto TY
agar plates containing 0.2 mM Dipy and 0.1 mM FeCl3 and then incubated at 28°C for
4 d. Cells spotted onto an TY agar plate (TY) were used as a control. (B) Cells were
grown in TY medium, TY medium containing 0.1 mM Dipy and TY medium
containing 0.1 mM Dipy and 0.1 mM FeCl3. Growth was monitored by measuring the
OD600 after incubation at 28°C with shaking for 36 h. Vertical bars indicate the
standard error for three independent experiments. An asterisk denotes a significant
difference from C58C1/vec in a t-test (* < 0.05 ).
Fig. 7. Transient transformation of tobacco leaves mediated by A. tumefaciens
strains. (A) Transient transformation of tobacco leaf cells by A. tumefaciens. Over 20
tobacco leaves were infiltrated with A. tumefaciens carrying pCAMBIA2301
construct. Bars, 1 mm. (B) and (C) Superoxide anion radical accumulation in tobacco
leaves stained with NBT after infiltration of H2O and suspensions of A. tumefaciens
cells, respectively, “ –” denotes an untreated leaf. These experiments were performed
three times. Over 20 tobacco leaves were infiltrated with A. tumefaciens strains. Bars,
1 mm. (D) Bacterial populations from infiltrated tobacco leaves. Vertical bars indicate
the standard error for three independent experiments. An asterisk denotes a significant
difference from C58C1 in a t-test (* < 0.05).
Fig. 8. Differential gene expression in the A. tumefaciens lsrB deletion mutant. A.
tumefaciens strains were cultured to OD600 = 0.5, and total RNA was extracted for
RNA-Seq and RT-qPCR. RNA-Seq was performed at Shanghai BoHao Corporation,
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Shanghai. Transcript levels of indicated genes were determined by RT-qPCR. Vertical
bars indicate the standard error for three independent experiments. The relative
amount of mRNA for each gene was calculated using the threshold cycle (∆Ct)
method, normalized to the ropD gene.
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The LsrB protein is required for Agrobacterium tumefaciens
interaction with host plants
Guirong Tang1, 2, Qiong Li1, Shenghui Xing1, Ningning Li1, Zheng Tang1,
Liangliang Yu1, Junhui Yan1, Xuan Li3, Li Luo1
1Shanghai Key Laboratory of Bio-energy Crops, Center of Plant Science, School of
Life Sciences, Shanghai University, Shanghai 200444, China; 2School of
Communication & Information Engineering, Shanghai University, Shanghai 200444,
China; 3Key Laboratory of Synthetic Biology, Institute of Plant Physiology and Ecology,
Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai
200032, China
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BrLsrB --MVAPLDWDKLRIFHAAAEAGSFTHAAQTLHLSQSAISRQVSALEQDVGVPLFHRHARG
SmLsrB MGDSMSLDWDKLRIFHAAAEAGSFTHAADKLHLSQSAISRQVSSLEQDVGIKLFHRHARG
AtLsrB --MAMPLDWDKLRIFHAAAEAGSFTHAADKLHLSQSAISRQVSALEQDVGVKLFHRHARG
**********************:.*************:******: ********
BrLsrB LILTEQGETLYRTAHDVLMKLENVRSKLAESREKPSGRLRVTTTVGLGSGWLIERIQEFV
SmLsrB LILTEQGEMLYRTAHDVLMKLESVKAQLSETTDKPSGKLRITTTVGLGQGWLTDKIQEFM
AtLsrB LILTEQGELLYRTAHDVLLKLETVKMQLTETTEKPSGKLRVTTTVGLGQGWLTDKVQEFL
******** *********:***.*: :*:*: :****:**:*******.*** :::***:
BrLsrB ELYPDVQLQLILDNEELDLTMRHADCAVRLRQPQQPDLIQRRLFIVHMHVYASAGYVSKY
SmLsrB SLYPEIQVQLILDNEELDVNMRHADCAIRLRQPQQSDLIQRKLFTVHMHVYAAPSYINKY
AtLsrB QLYPEMSIQLILDNEELDVNMRHADCAIRLRQPQQSDLIQRKLFTVHMHVYAAPSYINRH
.***::.:**********:.*******:******* *****:** *******: .*:.::
BrLsrB GKLNSIDEIDQHRIVTFGEPAPSYLTGLNWLETAGRPDGSARIPALQVNNLLSVRRAVQR
SmLsrB GEPQSLDDLDNHRIITFGEPAPNYLLDVNWLEIAGRDSDNPRISHLQINSQTSIKRACLL
AtLsrB GEPQSVEDLDNHRIISFGEPAPNYLLDVNWLENAGRSSDNTRIPHLQINSQTSIKRACLL
*: :*::::*:***::******.** .:**** *** ... ** **:*. *::**
BrLsrB GVGIAVLPDYMADKESGLVQLLPELEEIPSFDTFFCYPEALKNSAKLHAFRDFLFSKARN
SmLsrB GIGIAMLPDYIVGRDPGLIQL-PISADIPSFDTYFCYPDELKNAAKLKVFRDYIVAKARN
AtLsrB GIGIACLPDYIVGRDPGLIQL-SLAADIPSFDTYFCYPDEMKNAAKLKAFRDFIVAKARN
*:*** ****:..:: **:** :******:****: :**:***:.***::.:****
BrLsrB WTY
SmLsrB WNF
AtLsrB WNF
*.:
Fig. S1 Alignment of predicted LsrB proteins from Sinorhizobium meliloti,
Agrobacter tumefaciens and Brucella abortus. The amino acid sequences of LsrB
were available from NCBI. The CLUSTAL O was used for sequence alignment. The
DNA binding domains are shown as red parts.
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Fig. S2 Growth of A. tumefaciens lsrB mutants. Bacteria were cultured in LB/MC
medium. Cells in the exponential growth phase were diluted in fresh medium to
OD600=0.03. Cell density (optical density at 600 nm [OD600]) was monitored over time.
The figure shows the results of a typical experiment, which was performed three times.
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Fig. S3 Bacterial populations from infiltrated tobacco leaves. Vertical bars indicate the
standard error for three independent experiments. An asterisk denotes a significant
difference from C58C1 in a t-test (* < 0.05).
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Fig. S4 Location of lsrB and lrp3 genes in the genome of A. tumefaciens and S.
meliloti. The TN11A boxes recognized by LsrB were predicted in the promoter regions
of lrp3 genes.
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Table S1 Strains and plasmids
Strain or plasmid Relevant properties Reference/source
E.coli strains
DH5α F- supE44 ΔlacU169 (φ80lacZΔM15) hsdR17 (rK - mK
+ ) recA1 endA1
gyrA96 thi-1 relA1
TaKaRa Corp.
MT616 pro-82 thi-1 hsdR17 supE44 recA56(pRK600); CmR (Finan et al., 1984)
DA1 DH5α carring pA1 ; KmR This study
A.tumefaciens strains
C58C1 Agrobacterium rhizogens (StrR, RifR) pRiA4b (Jelenic et al., 2000)
∆lsrBat/ plsrBat ∆lsrBat carrying pSRK carrying A.tumefaciens strains C58C1 lsrB;
GmR
This study
∆lsrBat / plsrBsm ∆lsrBat carrying pSRK carrying S. meliloti 1021 lsrB; GmR This study
∆lsrBat / plsrBba ∆lsrBat carrying pSRK carrying B.abortus2308 lsrB; GmR This study
Plasmids
pK18mobsacB A suicide vector containing sacB; KmR (Schafer et al., 1994)
pA1 pK18mobsacB carrying lsrBat frank fragment ; KmR This study
plsrBsm pSRK carrying S. meliloti 1021 lsrB; GmR This study
plsrBba
plsrBat
pSRK
pHC60
pCAMBIA2301
pSRK carrying B.abortus 2308 lsrB; GmR
pSRK carrying A.tumefaciens C58C1 lsrB; GmR
Expressing vector under control of lac promoter; GmR
constitutive GFP construct
carrying a CAMV-35S driven-GUS gene
This study
This study
(Khan et al., 2008)
(Cheng and Walker,
1998)
(Cambia, Canberra,
Australia)
Reference
Cheng, H.P., and Walker, G.C. (1998) Succinoglycan is required for initiation and
elongation of infection threads during nodulation of alfalfa by Rhizobium meliloti. J
Bacteriol. 180: 5183-5191.
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Finan, T.M., Hartweig, E., LeMieux, K., Bergman, K., Walker, G.C., and Signer, E.R.
(1984) General transduction in Rhizobium meliloti. J Bacteriol 159: 120-124.
Jelenic, S., Mitrikeski, P.T., Papes, D., and Jelaska, S. (2000) Agrobacterium -
mediated transformation of broad bean Vicia faba L. Food Technol Biotech. 38: 167-
172.
Khan, S.R., Gaines, J., Roop, R.M., 2nd, and Farrand, S.K. (2008) Broad-host-range
expression vectors with tightly regulated promoters and their use to examine the
influence of TraR and TraM expression on Ti plasmid quorum sensing. Appl Environ
Microbiol 74: 5053-5062.
Schafer, A., Tauch, A., Jager, W., Kalinowski, J., Thierbach, G., and Puhler, A.
(1994) Small mobilizable multi-purpose cloning vectors derived from the Escherichia
coli plasmids pK18 and pK19: selection of defined deletions in the chromosome of
Corynebacterium glutamicum. Gene 145: 69-73.
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Table S2 Primers
Primers Sequence (5’ to 3’) Purpose
P1 GCTCTAGAGCTCACCGAAGTTGAGACCAA construction of pA1
P2 ATCTCGTCGGGATAGCAGAGCAGTTTATCCCAGTCCAA construction of pA1
P3 TCTGCTATCCCGACGAGAT construction of pA1
P4 CCCAAGCTTGGGATAATGGCGGAAGGCATC construction of pA1
P5 ACGAGCGACGAAGAGTCAT identify of ∆lsrBat
P6 CGGTGGATTATCTCAAGCG identify of ∆lsrBat
P7 GGGGTACCCACCGAAGTTGAAACGACT construction of plsrBba
P8 GCTCTAGACGTCAATAGGTCCAATTGCG construction of plsrBba
P9 GGGGTACCCC AAATCGTCAGCGACCTCGT construction of plsrBat
P10 GCTCTAGAGCCGTCAGAAGTTCCAGTTCCG construction of plsrBat
P11 GGGGTACCCCCGTGACCGACGACATTCAT construction of plsrBsm
P12 GCTCTAGATCAGAAGTTCCAGTTTCTC construction of plsrBsm
P13 AGACCATTTCCGCACATTG qRT-PCR of fecB
P14 TTGAAGACCGTGTTGTCGTTAG qRT-PCR of fecB
P15 CTTTCTGACCGCATTTGG qRT-PCR of fecD
P16 GCACGAGATTGACCTTCTGT qRT-PCR of fecD
P17 AACTGGCGGAAGGTGACAT qRT-PCR of gshA
P18 TTACCGAGGGCAAGCCTAA qRT-PCR of gshA
P29 CATCGCCGAAGAAGACAA qRT-PCR of katA
P20 CGAAGACATCAGAGAAATGGTC qRT-PCR of katA
P23 ACAAGAACGGCAACAAGG qRT-PCR of exoR
P24 CGGTAATAATCGGCAAGG qRT-PCR of exoR
P25 ATGCACAATGGATCAACG qRT-PCR of attD
P26 ACTGCACAATGACGGGTT qRT-PCR of attD
P27 ACCATTGCCGACACCTACA
qRT-PCR of ropD
P28 TTCAGCGACAGCGACTTGA qRT-PCR of ropD
P29 GCCCACCAGATTGAAATACT qRT-PCR of fecE
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P30
P31
P32
P32
P34
P35
P36
P37
P38
P39
P40
P41
P42
P43
P44
P45
P46
P47
P48
GTTGACGCCAAAGACATCA
TCCAGAATACCGAAGACCTG
TTTCCACCGTGAACGAAC
TTCACGCTGCATTATCCC
ACCGTCAAACTGGGCTTT
AGCGGTTATGGTTATGGC
ACCTTTGAAGTCTGGCATCT
CAGTCTGTCAGCAAGAACCAG
ATCGGCAACGGAAATCAG
CGAAGCCTGCGATTACAT
TGTCGGTCTTGTTGAGCA
CCGAAGCAAACAGGGAAT
TTCATTTCACGCCAGACC
GAGCAAGGAAACCAATCTCA
ATGAAGAACCGCACATCG
GCTGGCAAGGTCAAAGTT
CTTCGCCACCCTTGAATA
TGGAAGAAGTGCCGCTAT
AACTGCCTTCCATCAGCA
qRT-PCR of fecE
qRT-PCR of Atu3676
qRT-PCR of Atu3676
qRT-PCR of Atu3679
qRT-PCR of Atu3679
qRT-PCR of exoV
qRT-PCR of exoV
qRT-PCR of exoC
qRT-PCR of exoC
qRT-PCR of exoP
qRT-PCR of exoP
qRT-PCR of Atu3680
qRT-PCR of Atu3680
qRT-PCR of gshB
qRT-PCR of gshB
qRT-PCR of trxA
qRT-PCR of trxA
qRT-PCR of oxyR
qRT-PCR of oxyR
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Table S3 Gene differential expression in the lsrB deletion mutant
gene id gene
name description
mutant/wt
Change fold
(log2)
P value
1132315 - hypothetical_protein -7.78836993 0
1133705 - hypothetical_protein -7.59033078 0
1133069 - hypothetical_protein -6.15964948 0
1139135 - hypothetical_protein -5.08156645 0
1134968 scrK fructokinase -4.90097779 0
1134969 agaZ tagatose_6_phosphate_kinase -4.82562762 0
1134966 - sorbitol_dehydrogenase -4.66561167 0
1134965 - zinc_binding_dehydrogenase -4.32933555 0
1135735 - acyl_CoA_hydrolase -4.28343574 3.8853E-269
1136415 pcaC carboxymuconolactone_decarboxylase -4.22011601 6.87849E-79
1136594 dppB dipeptide_ABC_transporter_permease -4.17502511 7.8873E-179
1134967 - sorbitol_mannitol_ABC_transporter_substrate_binding_protein -4.06906458 0
1136595 dppC dipeptide_ABC_transporter_permease -4.03868529 2.3658E-154
1136422 pcaJ 3_oxoadipate_CoA_transferase_subunit_B -3.98938885 1.2531E-135
1136421 pcaI 3_oxoadipate_CoA_transferase_subunit_A -3.85514944 2.31331E-94
1137500 virB11 type_IV_secretion_system_protein_VirB11 -3.82461734 0.003513922
1136596 ggt gamma_glutamyltranspeptidase -3.697382 4.94222462277237e-
317
1134972 - sorbitol_mannitol_ABC_transporter_ATPase -3.6283131 0
1136423 pcaF beta_ketoadipyl_CoA_thiolase -3.51043545 3.9565E-182
1133070 - hypothetical_protein -3.46447239 0
1136593 dppA dipeptide_ABC_transporter_substrate_binding_protein -3.29517148 3.3206E-242
1136592 - GntR_family_transcriptional_regulator -3.22545492 1.12533E-52
1136416 pcaD beta_ketoadipate_enol_lactone_hydrolase -3.21632437 2.89927E-52
1134224 - transcriptional_regulator%2C_LysR_family -3.1874254 0
1134971 - sorbitol_mannitol_ABC_transporter_permease -2.9799745 1.1644E-223
1134970 - sorbitol_mannitol_ABC_transporter_permease -2.91080598 2.1841E-226
1135918 - glycine_betaine_ABC_transporter_ATPase -2.90456326 4.6379E-297
1136597 - ABC_transporter_permease -2.8806064 2.456E-246
1133802 metF methylenetetrahydrofolate_reductase -2.73893103 5.5721E-256
1134553 dppD ABC_transporter%2C_nucleotide_binding_ATPase_protein_dipeptide_ -2.73407391 2.7125E-153
1135697 metE 5_methyltetrahydropteroyltriglutamate_homocysteine_S_methyltransferase -2.6952744 1.8015E-285
1133123 slyA transcriptional_regulator%2C_MarR_family -2.59373547 6.97114E-81
1136389 livF branched_chain_amino_acid_ABC_transporter_ATPase -2.556168 4.0773E-107
1136896 atrC acetolactate_synthase_catalytic_subunit -2.5285444 1.5997E-257
1136897 atrB glutamate_1_semialdehyde_aminotransferase -2.50879528 3.0299E-241
1132764 - ring_hydroxylating_dioxygenase%2C_alpha_subunit -2.50575923 2.5759E-270
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1133670 - dimethylglycine_dehydrogenase -2.5045827 1.1754E-270
1134180 glnA glutamine_synthetase -2.48454783 1.0528E-182
1134554 dppC ABC_transporter%2C_membrane_spanning_protein_dipeptide_ -2.48239288 6.5038E-140
1134551 - penicillin_binding_protein -2.45473202 7.0796E-109
1136412 pcaB 3_carboxy_cis%2Ccis_muconate_cycloisomerase -2.44415127 4.1854E-135
1135883 tnp IS3_family_transposase -2.44070213 0.000595325
1132993 - hypothetical_protein -2.37025666 5.82546E-13
1136414 pcaH protocatechuate_3%2C4_dioxygenase_subunit_beta -2.36252019 1.4859E-127
1134552 dppF ABC_transporter%2C_nucleotide_binding_ATPase_protein_dipeptide_ -2.35762433 3.0215E-104
1136390 livG branched_chain_amino_acid_ABC_transporter_ATPase -2.34103367 2.43855E-80
1135698 - hypothetical_protein -2.33450449 3.2714E-198
1135943 soxA sarcosine_oxidase_alpha_subunit -2.32652645 1.6854E-228
1135944 soxG sarcosine_oxidase_gamma_subunit -2.31622832 1.2368E-187
1137288 - hypothetical_protein -2.29542076 9.15534E-33
1136413 pcaG protocatechuate_3%2C4_dioxygenase_alpha_chain -2.28466748 1.9851E-107
1134556 dppA ABC_transporter%2C_substrate_binding_protein_dipeptide_ -2.27543127 1.1697E-209
1134548 - aminopeptidase -2.2457405 8.283E-135
1132483 - hypothetical_protein -2.20057482 1.31933E-17
1136902 attC ABC_transporter_substrate_binding_protein_mannopine_ -2.19678281 6.1174E-174
1137497 virB8 type_IV_secretion_system_protein_VirB8 -2.17776098 0.001546551
1132765 - ferredoxin_I -2.14059824 8.4603E-195
1134974 - sugar_ABC_transporter_permease -2.12578172 5.09486E-49
1136058 - - -2.12304152 2.39193E-66
1136895 fabG 3_ketoacyl_ACP_reductase -2.11876152 9.3269E-129
1136894 - two_component_response_regulator -2.11490445 1.65068E-74
1136903 attD attachment_protein -2.10441154 1.64917E-56
1136583 fdsD NAD_dependent_formate_dehydrogenase_subunit_delta -2.09702018 4.53876E-96
1134555 dppB ABC_transporter%2C_membrane_spanning_protein_dipeptide_ -2.05033517 2.7618E-135
1137499 virB10 type_IV_secretion_system_protein_VirB10 -2.04638992 7.83555E-05
1132103 frcA ABC_transporter%2C_nucleotide_binding_ATPase_protein_sugar_ -2.04229486 5.5793E-181
1132484 - hypothetical_protein -2.03259189 2.00847E-21
1132994 gp35 phage_prohead_protease -1.99391887 7.83273E-12
1132104 - putative_kinase -1.97833913 1.6306E-155
1135920 - glycine_betaine_ABC_transporter_substrate_binding_protein -1.94043105 4.4122E-161
1135919 - glycine_betaine_ABC_transporter_permease -1.92650647 4.5429E-134
1134973 - sugar_ABC_transporter_ATPase -1.90913111 4.20207E-65
1136184 soxB sarcosine_oxidase_beta_subunit -1.90121826 1.43837E-69
1132867 betB betaine_aldehyde_dehydrogenase -1.88342844 6.73053E-81
1135359 - short_chain_dehydrogenase -1.87601489 3.12572E-89
1132868 betA choline_dehydrogenase -1.86852138 3.93781E-63
1134061 ndh NADH_dehydrogenase -1.86317315 1.0512E-122
1132866 betI transcriptional_regulator_BetI -1.86235001 9.03215E-26
1135358 - hypothetical_protein -1.85258879 1.23641E-73
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or p
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. The
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fer.
1136391 livM branched_chain_amino_acid_ABC_transporter_permease -1.82571018 3.08579E-52
1135767 - sugarl_ABC_transporter_permease -1.80590922 2.62867E-42
1132632 aglA alpha_glucosidase -1.79010837 1.3245E-142
1132500 - endolysin -1.77633566 9.30458E-57
1132633 aglK ABC_transporter%2C_nucleotide_binding_ATPase_protein -1.76499557 1.2686E-138
1132502 - hypothetical_protein -1.74698297 7.14271E-24
1135667 aspA aspartate_ammonia_lyase -1.74633339 8.79235E-51
1137289 - hypothetical_protein -1.74329647 8.33975E-80
1136901 attB ABC_transporter_membrane_spanning_protein_mannopine_ -1.74275 1.82072E-76
1136185 soxD sarcosine_oxidase_delta_subunit -1.7187827 5.31697E-23
1135768 - sugar_ABC_transporter_permease -1.70676402 4.83284E-36
1134182 - hypothetical_protein -1.70439161 2.812E-40
1134502 purU formyltetrahydrofolate_deformylase -1.67733192 4.879E-114
1136582 fdhF formate_dehydrogenase_alpha_subunit -1.67678739 3.2402E-116
1136666 - hypothetical_protein -1.66968974 3.42048E-06
1135561 - TonB_dependent_receptor -1.66538822 2.3806E-103
1137958 - hypothetical_protein -1.65986795 1.4323E-07
1136186 soxA sarcosine_oxidase_alpha_subunit -1.65225556 1.46521E-88
1135885 - hypothetical_protein -1.64205033 3.24789E-38
1134188 - ABC_transporter%2C_nucleotide_binding_ATPase_protein -1.64089172 5.37668E-87
1134181 - ABC_transporter%2C_membrane_spanning_protein_amino_acid_ -1.6358321 4.99528E-60
1136899 attA1 ABC_transporter_nucleotide_binding_ATPase_putrescine_ -1.63465103 4.97092E-88
1135365 - hypothetical_protein -1.63311097 2.44066E-15
1136584 - MFS_permease -1.62076846 1.7353E-101
1134975 - sugar_ABC_transporter_permease -1.61974397 2.49763E-31
1135942 soxD sarcosine_oxidase_delta_subunit -1.61921802 8.63007E-71
1132995 gp36 phage_phi_C31_major_capsid_gp36_like_protein -1.61064832 3.161E-38
1136900 attA2 ABC_transporter_membrane_spanning_protein_mannopine_ -1.60944414 6.70496E-79
1133004 - hypothetical_protein -1.60160353 5.43429E-09
1133605 fdhA glutathione_independent_formaldehyde_dehydrogenase -1.59821899 4.4023E-110
1133002 - hypothetical_protein -1.59346854 1.24087E-14
1132802 - hypothetical_protein -1.58568523 2.20962E-94
1134964 - LacI_family_transcriptional_regulator -1.57691751 1.19907E-83
1136985 - hypothetical_protein -1.57253765 4.50621E-38
1135941 - hypothetical_protein -1.568207 1.57548E-78
1137970 - hypothetical_protein -1.56550462 2.66714E-79
1132499 - hypothetical_protein -1.55908685 2.5699E-48
1135562 fecB iron_III_ABC_transporter_permease_dicitrate_ABC_transporter_permease -1.55362373 1.72074E-54
1135565 fecE iron_III_dicitrate_ABC_transporter_ATPase -1.55131628 3.18756E-26
1135947 glgX glycogen_debranching_protein -1.54573059 6.22794E-85
1132631 aglG ABC_transporter%2C_membrane_spanning_protein -1.53629522 2.3942E-103
1136910 blcA NAD_dependent_succinyl_semialdehyde_dehydrogenase -1.52454302 2.28979E-57
1138472 - hypothetical_protein -1.5162651 1.28803E-42
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1135924 exoN UTP_glucose_1_phosphate_uridylyltransferase -1.5158058 1.56292E-98
1132766 - hypothetical_protein -1.50878312 6.47472E-56
1136189 purU formyltetrahydrofolate_deformylase -1.50815479 6.63079E-28
1133805 - transcriptional_regulator%2C_AraC_family -1.50368363 1.19469E-93
1133447 allA ureidoglycolate_hydrolase -1.49926947 1.04267E-13
1136418 pobA 4_hydroxybenzoate_3_monooxygenase -1.49797762 1.83517E-26
1134123 lpxC UDP_3_0_3_hydroxymyristoyl_N_acetylglucosamine_deacetylase -1.49224529 2.40576E-83
1136893 - hypothetical_protein -1.48414311 2.10793E-05
1136187 soxG sarcosine_oxidase_gamma_subunit -1.47283421 2.03491E-26
1132801 - hydrolase -1.47082915 4.0974E-75
1137969 - putative_phage_tail_protein_I -1.46703397 2.48884E-29
1135940 soxB sarcosine_oxidase_beta_subunit -1.45039662 3.04408E-87
1132492 - hypothetical_protein -1.44225099 7.44952E-27
1135769 - sugar_ABC_transporter_ATPase -1.4392144 6.09735E-25
1132482 - hypothetical_protein -1.43834167 4.35522E-05
1132932 - ABC_transporter%2C_membrane_spanning_protein -1.43163834 0.000547753
1137963 - hypothetical_protein -1.42789553 1.07785E-29
1136614 - zinc_binding_dehydrogenase -1.4223455 4.49176E-19
1132506 - hypothetical_protein -1.41728873 1.17844E-50
1135541 panD aspartate_alpha_decarboxylase -1.4120448 3.11845E-23
1137449 traC conjugal_transfer_protein_TraC -1.40504158 1.26288E-06
1132997 - hypothetical_protein -1.39949776 1.59941E-05
1137756 - hypothetical_protein -1.39858357 9.97688E-64
1134525 pdxK pyridoxamine_kinase -1.39313489 1.12907E-65
1133124 - MFS_permease -1.39291231 1.58621E-37
1135948 exoC phosphoglucomutase -1.38564463 1.29932E-81
1137973 - putative_bacteriophage_P2_tail_protein -1.38447075 2.63194E-21
1132495 - hypothetical_protein -1.38415034 9.91509E-55
1134383 gguC hypothetical_protein -1.38368142 1.37294E-80
1137946 - hypothetical_protein -1.38213962 1.62594E-20
1135772 glpR DeoR_family_transcriptional_regulator -1.37282902 9.27793E-27
1133445 - gluconate_dehydrogenase -1.37028106 5.36241E-36
1136841 - ABC_transporter_nucleotide_binding_ATPase_oligopeptide_ -1.36804827 2.15137E-41
1135366 - sugar_kinase -1.3593303 1.39033E-28
1134179 - hypothetical_protein -1.35323732 4.29407E-58
1134178 - hypothetical_protein -1.35170011 1.59734E-47
1135701 - LysR_family_transcriptional_regulator -1.34843307 2.04876E-55
1133496 mvrA ferredoxin_NADP+_reductase -1.34501453 1.93538E-70
1133003 - hypothetical_protein -1.34383749 5.00414E-16
1133446 gutB sorbitol_dehydrogenase -1.34103535 6.19463E-25
1135766 - hypothetical_protein -1.34012556 3.34013E-08
1137950 - hypothetical_protein -1.33972698 3.1997E-10
1132630 aglF ABC_transporter%2C_membrane_spanning_protein -1.33522008 6.64794E-78
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1135923 exoP exopolysaccharide_polymerization_transport_protein -1.33178598 3.60061E-76
1136826 - hypothetical_protein -1.32901695 1.01342E-11
1136236 - hypothetical_protein -1.3284458 4.39335E-26
1136232 - ABC_transporter_permease -1.31860459 5.63567E-25
1132491 - hypothetical_protein -1.3163302 2.19225E-31
1135564 fecD iron_III_ABC_transporter_permease_dicitrate_ABC_transporter_permease -1.31620349 6.53524E-18
1135950 glgC glucose_1_phosphate_adenylyltransferase -1.30531202 1.83859E-69
1136382 - oxoreductase -1.30477837 7.69738E-49
1136278 - hypothetical_protein -1.30236041 3.58145E-09
1133797 sdaA L_serine_dehydratase -1.30201465 4.0133E-72
1137964 - hypothetical_protein -1.29648872 1.72053E-25
1132487 - hypothetical_protein -1.2952575 4.55555E-15
1136420 pcaR IclR_family_transcriptional_regulator -1.29417446 2.21141E-15
1136939 avhB5 type_IV_secretion_protein_AvhB5 -1.29270779 1.53935E-26
1137234 - ABC_transporter_membrane_spanning_protein -1.29264089 3.86236E-25
1137272 - oxidoreductase -1.29171871 7.87108E-13
1136883 traC conjugal_transfer_protein -1.291508 1.04716E-06
1134976 - sugar_ABC_transporter_substrate_binding_protein -1.29034965 6.00906E-53
1136840 dapA dihydrodipicolinate_synthase -1.28790378 5.67758E-35
1136392 livH branched_chain_amino_acid_ABC_transporter_permease -1.28698975 2.61664E-35
1132073 pckA phosphoenolpyruvate_carboxykinase -1.28631229 4.43621E-62
1138454 - hypothetical_protein -1.28192763 4.89949E-12
1134187 - ABC_transporter%2C_membrane_spanning_protein -1.28024682 2.57381E-39
1137366 repA plasmid_partitioning_protein_RepA -1.27857546 0.005137498
1136938 avhB4 type_IV_secretion_protein_AvhB4 -1.2751934 5.75068E-43
1134183 - transcriptional_regulator%2C_RpiR_family -1.2720662 5.87477E-31
1132989 - large_terminase_phage_packaging_protein -1.26890979 4.2871E-20
1132992 gp34 phage_head_portal_protein -1.26535183 4.3801E-15
1136383 iolB hypothetical_protein -1.26005091 7.58523E-53
1136825 hspAT1 small_heat_shock_protein -1.25412206 2.92351E-24
1132485 - hypothetical_protein -1.25210111 2.26664E-06
1136188 glyA serine_hydroxymethyltransferase -1.25168693 4.94496E-44
1133565 fixS nitrogen_fixation_protein_FixS -1.24742775 1.38086E-33
1132534 - hypothetical_protein -1.24482011 2.783E-30
1135540 - hypothetical_protein -1.23664575 4.34943E-22
1137235 - ABC_transporter_nucleotide_binding_ATPase -1.23301341 5.49332E-29
1135743 - ABC_transporter_permease -1.23262632 9.25581E-60
1133467 pdhA pyruvate_dehydrogenase_alpha_subunit -1.23183132 1.25939E-66
1137951 - hypothetical_protein -1.22983736 1.98794E-18
1136360 - nitroreductase -1.22390927 6.39583E-34
1133669 rrpX two_component_response_regulator -1.22379276 7.04185E-47
1135550 Atu3676 siderophore_biosynthesis_protein -1.22089922 3.81454E-08
1134777 - oxidoreductase -1.2165309 3.83002E-22
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1137270 - hypothetical_protein -1.21207102 3.38431E-10
1133030 - permease -1.21155448 1.38197E-13
1134634 - beta_N_acetylhexosaminidase -1.21150294 7.27789E-52
1138452 - hypothetical_protein -1.18971645 1.97125E-10
1137273 - dehydrogenase -1.18636849 3.68278E-28
1132933 - ABC_transporter%2C_substrate_binding_protein -1.18407438 1.1289E-09
1135553 Atu3679 siderophore_biosynthesis_protein -1.18246507 1.4882E-06
1136564 - amino_acid_ABC_transporter_ATPase_permease -1.17650445 2.88286E-28
1133617 - ABC_transporter%2C_membrane_spanning_protein_amino_acid_ -1.17531919 3.92743E-58
1139497 - hypothetical_protein -1.17181624 1.38609E-56
1133473 - hypothetical_protein -1.17114156 1.04358E-47
1135005 - RND_multidrug_efflux_membrane_permease -1.16897858 2.94744E-30
1136524 - sugar_ABC_transporter_ATPase -1.16291872 5.01154E-28
1135949 glgA glycogen_synthase -1.16019481 9.42834E-54
1136911 blcB gamma_hydroxybutyrate_dehydrogenase -1.15969417 2.09551E-34
1136723 - NADP_dependent_aldehyde_dehydrogenase -1.15933732 6.84326E-50
1134549 dat D_alanine_aminotransferase -1.15847611 8.75046E-39
1134176 - dehydrogenase -1.15643601 2.57445E-54
1136937 avhB3 type_IV_secretion_protein_AvhB3 -1.1526202 1.05177E-12
1136722 deoC 2_deoxyribose_5_phosphate_aldolase -1.15172615 1.18997E-38
1136941 avhB7 type_IV_secretion_protein_AvhB7 -1.14852095 8.30139E-07
1135363 - ABC_transporter_permease -1.14732163 1.48768E-17
1135554 Atu3680 siderophore_biosynthesis_protein -1.14593126 0.000379359
1135563 fecC iron_III_ABC_transporter_permease_dicitrate_ABC_transporter_permease -1.14394468 5.34493E-16
1135688 - hypothetical_protein -1.14118846 8.35863E-06
1132629 aglE ABC_transporter%2C_substrate_binding_protein_alpha_glucoside_ -1.1342628 2.00748E-57
1136003 mmsB 3_hydroxyisobutyrate_dehydrogenase -1.1299013 1.04029E-19
1136537 - oligopeptide_ABC_transporter_permease -1.12792667 3.16187E-29
1135091 aspC aspartate_aminotransferase -1.12549699 2.51357E-21
1136630 - hypothetical_protein -1.12430697 2.63432E-15
1136525 argH argininosuccinate_lyase -1.12319698 7.94902E-28
1134983 - hypothetical_protein -1.12166858 0.001425402
1134287 - hypothetical_protein -1.11769511 3.60598E-28
1135770 - sugar_ABC_transporter_ATPase -1.11695295 1.24816E-14
1134716 ligT 2_5_RNA_ligase -1.11509824 1.46421E-49
1132511 - hypothetical_protein -1.11494739 2.03273E-12
1135967 - hypothetical_protein -1.111456 2.91893E-29
1135552 Atu3678 siderophore_biosynthesis_protein -1.11071039 0.000144725
1135201 exoY succinoglycan_exopolysaccharide_synthesis_protein -1.10989497 3.96119E-52
1135004 - RND_multidrug_efflux_transporter -1.1051447 2.28077E-40
1136384 iolE hypothetical_protein -1.09964375 5.09681E-42
1134009 - hypothetical_protein -1.09424924 1.16024E-45
1135933 exoV succinoglycan_biosynthesis_protein -1.0880879 1.15341E-39
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1132486 - hypothetical_protein -1.0870508 0.017888366
1136943 avhB9 type_IV_secretion_protein_AvhB9 -1.08637926 2.73208E-14
1134177 - aldehyde_dehydrogenase -1.08598679 4.28646E-46
1134437 - hypothetical_protein -1.08126895 1.15042E-44
1140140 pycA pyruvate_carboxylase -1.07732537 1.13345E-50
1132488 nusG transcription_antitermination_protein -1.076038 2.46699E-08
1133572 fixP cytochrome_c_oxidase%2C_FixP_chain -1.07577354 2.18524E-49
1135736 - hypothetical_protein -1.0749841 2.00647E-22
1133443 - transcriptional_regulator%2C_GntR_family -1.07438286 4.05813E-34
1134635 - short_chain_dehydrogenase -1.07323507 1.59578E-25
5729694 - hypothetical_protein -1.07088319 1.13264E-35
1136944 avhB10 type_IV_secretion_protein_AvhB10 -1.06804906 1.30351E-19
1138468 - hypothetical_protein -1.06766535 1.8105E-08
1137231 - isomerase_lactonizing_enzyme -1.06645835 1.09722E-21
1136393 livJ branched_amino_acid_ABC_transporter_substrate_binding_protein -1.06293261 9.84163E-36
1136936 avhB2 type_IV_secretion_protein_AvhB2 -1.06282937 2.19485E-11
1135702 - acetyltransferase -1.06235437 2.81131E-24
1133618 - ABC_transporter%2C_nucleotide_binding_ATPase_protein_amino_acid_ -1.06182612 2.87663E-47
1137233 - ABC_transporter_membrane_spanning_protein -1.05477153 6.39188E-19
1136880 - hypothetical_protein -1.05346922 6.93329E-05
1135969 - hypothetical_protein -1.05148098 2.88405E-22
1132498 - hypothetical_protein -1.04311442 5.47423E-05
1135691 - dehydratase -1.04109584 2.5328E-15
1136005 - hydroxybutyrate_dehydrogenase -1.03906738 2.67309E-14
1136118 - polyamine_ABC_transporter_permease -1.03356812 1.80004E-07
1132650 - ABC_transporter%2C_membrane_spanning_protein -1.03219426 5.62992E-26
1136114 - alkanal_monooxygenase_subunit_alpha -1.03210872 2.01935E-12
1136882 traD conjugal_transfer_protein -1.03121922 0.001720061
1135549 - peptide_synthetase%2C_siderophore_biosynthesis_protein -1.02951634 5.71903E-16
1135216 thuA trehalose_utilization_like_protein -1.02934362 5.72771E-43
1137229 - hypothetical_protein -1.0249852 5.36614E-17
1134633 - hypothetical_protein -1.02381235 1.39607E-34
1132102 frcC ABC_transporter%2C_membrane_spanning_protein_sugar_ -1.02377531 7.665E-46
1134774 ilvD dihydroxy_acid_dehydratase -1.02119187 2.21128E-26
1133472 lpdA dihydrolipoamide_dehydrogenase -1.01666482 1.2033E-43
1134175 - hypothetical_protein -1.01643 1.05775E-32
1136208 impJ hypothetical_protein -1.01620593 2.17112E-40
1135090 - oligopeptide_ABC_transporter_ATPase -1.01433479 1.09606E-06
1136604 - hypothetical_protein -1.01373878 8.95258E-06
1137271 - oxidoreductase_with_iron_sulfur_subunit -1.01163914 0.000644486
1135337 cpoF non_heme_chloroperoxidase -1.0107668 0.002245145
1136633 eutB threonine_dehydratase -1.00995016 1.31584E-07
1135200 exoF exopolysaccharide_production_protein -1.00871563 2.25017E-39
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1135215 thuK trehalose_maltose_ABC_transporter_ATPase -1.00635381 5.43746E-37
1136538 - oligopeptide_ABC_transporter_ATPase -1.00553412 2.53735E-30
1134896 mcpA methyl_accepting_chemotaxis_protein -1.00403787 2.831E-14
1135219 exsC 6_pyruvoyl_tetrahydrobiopterin_synthase -1.00260888 0.001457933
1135413 - hypothetical_protein -1.0022391 1.36705E-37
1134439 ureC urease_alpha_subunit -1.00160009 1.29037E-37
1133006 - hypothetical_protein -1.00042329 3.94988E-09
1135833 dhaL aldehyde_dehydrogenase -1.00031244 1.60272E-22
1134568 - oxidoreductase -1.01056915 5.47839E-09
1134371 - ATP_dependent_RNA_helicase 1.013705246 1.58513E-56
1136656 drrA two_component_response_regulator 1.032536482 1.67049E-36
1133500 gcvP glycine_cleavage_system_protein_P2 1.033741038 7.54667E-65
1134425 - NTP_pyrophosphohydrolase%2C_MutT_family 1.034719339 4.82761E-17
1136290 - phosphopantetheinyl_transferase 1.043450021 6.38702E-51
1134050 - ABC_transporter%2C_nucleotide_binding_ATPase_protein 1.0579565 7.33563E-05
1134423 amaB N_carbamoyl_beta_alanine_amidohydrolase 1.059826536 2.1631E-43
1134471 - hypothetical_protein 1.060540569 0.017288282
1132427 - - 1.064746856 4.27506E-13
1134565 - dioxygenase 1.071734803 1.71222E-06
1134106 - hypothetical_protein 1.072100393 5.2714E-37
1134426 - ABC_transporter%2C_nucleotide_binding_ATPase_protein_nitrate_ 1.073551288 2.33514E-24
1133755 fadL long_chain_fatty_acid_transport_protein 1.077526427 2.78574E-68
1134820 - acetyltransferase 1.077778581 1.31711E-53
1136024 - hypothetical_protein 1.078471698 5.30887E-37
1134937 - ABC_transporter_permease 1.082887735 1.59188E-15
1132543 - - 1.087830166 6.61109E-17
1133099 - hypothetical_protein 1.089220702 1.72595E-17
1135879 - - 1.099275763 1.20728E-08
1134950 - sugar_ABC_transporter_ATPase 1.102809053 2.10261E-18
1134424 dht dihydropyrimidinase 1.111738073 4.0744E-43
1135619 - FAD_binding_dehydrogenase 1.123212042 9.08813E-53
1133835 - - 1.129496549 9.93185E-09
1134949 - oxidoreductase 1.137909701 1.28387E-42
1135112 - sugar_ABC_transporter_permease 1.139913534 6.16285E-24
1133606 - transcriptional_regulator%2C_TetR_family 1.140413415 1.31436E-29
1134451 - ABC_transporter%2C_membrane_spanning_protein_urea_amide_ 1.143460598 0.000115277
1134534 - nitroreducatase 1.144430407 7.93655E-37
1135783 - hypothetical_protein 1.155340997 5.10517E-75
1136028 - aliphatic_sulfonate_ABC_transporter_substrate_binding_protein 1.155822605 3.45792E-20
1133621 - - 1.160365971 2.70464E-08
1136400 - ABC_transporter_permease 1.160965037 3.97535E-75
1135872 bioF 8_amino_7_oxononanoate_synthase 1.170115549 5.48999E-35
1136655 - two_component_sensor_kinase 1.170386966 1.53014E-38
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1133501 gcvH glycine_cleavage_system_component_H 1.173994553 1.12185E-68
1135347 bkdA1 2_oxoisovalerate_dehydrogenase_subunit_alpha 1.178147254 1.02606E-76
1135172 dctA C4_dicarboxylate_transporter_DctA 1.182580635 1.03711E-72
1135954 - glutamine_amidotransferase 1.183193994 1.01501E-40
1136295 - spermidine_putrescine_ABC_transporter_substrate_binding_protein 1.184188456 1.16287E-75
1135129 - dicarboxylate_ABC_transporter_permease 1.184920909 3.00314E-61
1134916 - sugar_ABC_transporter_substrate_binding_protein 1.185905855 3.54537E-52
1134262 aldA aldehyde_dehydrogenase 1.19648823 2.86052E-83
1134341 - hypothetical_protein 1.197805395 1.5434E-44
1132153 - hypothetical_protein 1.201528094 1.00586E-14
1132501 - hypothetical_protein 1.20574237 0.000914969
1135126 exuR GntR_family_transcriptional_regulator 1.209106849 4.83877E-33
1135351 acd acyl_CoA_dehydrogenase 1.210722394 5.35136E-78
1133024 - hypothetical_protein 1.214667461 0.000251116
1137257 - hypothetical_protein 1.217068797 8.66712E-73
1132066 - hypothetical_protein 1.218183039 2.67732E-09
1136299 iunH inosine_uridine_preferring_nucleoside_hydrolase 1.225343827 2.48424E-30
1136452 - ABC_transporter_permease 1.237930691 7.77898E-57
1136696 - acyl_CoA_synthetase 1.24542892 6.53728E-72
1134429 - ABC_transporter%2C_substrate_binding_protein_nitrate_sulfonate_taurine_bicarbonate_ 1.25120185 1.82699E-46
1136697 acaB acetyl_CoA_acetyltransferase 1.253172143 8.89726E-68
1132451 - stress_induced_protein 1.253997163 2.46233E-05
1132328 - rare_lipoprotein_A 1.256515934 1.49416E-49
1133303 - - 1.262153438 1.27851E-10
1132891 - hypothetical_protein 1.272918244 1.92047E-35
1132337 - hypothetical_protein 1.292821731 8.6763E-57
1136695 fabG 3_oxoacyl_ACP_reductase 1.29404792 6.31029E-56
1136694 - hypothetical_protein 1.298250872 2.64746E-49
1133593 - - 1.304968825 9.06443E-20
1134938 - ABC_transporter_permease 1.311164098 3.35027E-08
1132522 - two_component_response_regulator 1.312340127 7.04779E-81
1135077 - LacI_family_transcriptional_regulator 1.313256349 1.0029E-86
1135226 - sugar_ABC_transporter_substrate_binding_protein 1.3369134 6.69824E-79
1136059 - hypothetical_protein 1.337504513 2.13004E-81
1135240 - tripartite_ATP_independent_periplasmic_transporter_DctQ 1.339624179 1.51641E-54
1135574 - - 1.345928458 4.33951E-23
1135078 - ABC_transporter_permease 1.34981007 1.63321E-95
1132771 - enoyl_CoA_hydratase_isomerase 1.351071395 3.51722E-57
1134273 - hypothetical_protein 1.352464121 2.05712E-82
1135976 - - 1.353011049 2.92569E-29
1132196 - ABC_transporter%2C_membrane_spanning_protein 1.354516115 5.3486E-85
1137043 - permease_component_of_C4_dicarboxylate_transporter 1.370012785 4.0654E-100
1134987 ugpB glycerol_3_phosphate_ABC_transporter_substrate_binding_protein 1.380162044 1.0513E-88
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1134535 - hydrolase 1.384080745 2.59759E-65
1133231 - - 1.398366038 0.008189464
1135431 - hypothetical_protein 1.39838269 2.00436E-06
1135407 - sugar_ABC_transporter_substrate_binding_protein 1.398982826 6.7909E-72
1135818 - - 1.413047331 2.03868E-09
1136698 - IclR_family_transcriptional_regulator 1.418515094 1.41369E-65
1141089 - hypothetical_protein 1.422663447 7.57713E-97
1136297 - ABC_transporter_permease 1.423640425 8.36515E-39
1133623 - - 1.423909312 2.21234E-07
1135146 pssN exopolysaccharide_export_protein 1.428861112 2.32856E-45
1133259 - hypothetical_protein 1.453033113 3.92742E-60
1134577 - - 1.455638045 2.56101E-25
1136300 adeC adenine_deaminase 1.459903247 8.40018E-60
1136658 afuA ABC_transporter_substrate_binding_protein 1.470925571 5.86838E-45
1134536 - hypothetical_protein 1.478612962 5.40148E-53
1134272 - hypothetical_protein 1.486804266 7.8569E-101
1135020 - hypothetical_protein 1.497911703 0.003687528
1132098 - - 1.503478538 3.42845E-07
1136580 nuoE formate_dehydrogenase_subunit_gamma 1.527344469 1.53059E-92
1136701 - ABC_transporter_permease 1.528395791 4.9333E-107
1135882 - - 1.541486662 8.48069E-98
1134743 prsD1 ABC_transporter%2C_nucleotide_binding_ATPase_protein_protein_ 1.54583836 3.5178E-42
1135068 - peptide_ABC_transporter_ATPase 1.555717027 3.39171E-97
1136296 - spermidine_putrescine_ABC_transporter_permease 1.57109518 1.69181E-84
1134537 - Isochorismatase 1.579096898 1.42664E-85
1135167 dadA D_amino_acid_dehydrogenase_small_subunit 1.580557155 1.7791E-132
1135907 - sugar_ABC_transporter_substrate_binding_protein 1.593893868 1.3993E-141
1136066 - oligopeptide_ABC_transporter_substrate_binding_protein 1.59617217 3.7832E-113
1135348 mmgC acyl_CoA_dehydrogenase 1.62431202 1.4798E-137
1135881 arcA arginase 1.625550034 6.6512E-126
1136618 - sugar_ABC_transporter_substrate_binding_protein 1.633283722 4.9271E-81
1134150 slt soluble_lytic_transglycosylase 1.637524772 6.0681E-31
1135002 - ribose_ABC_transporter_ATPase 1.64186862 1.1506E-105
1132145 - hypothetical_protein 1.674921574 5.9525E-103
1134006 - hypothetical_protein 1.699616069 0.008583897
1136699 - dehydrogenase 1.711517521 7.521E-114
1135664 kdpA potassium_transporting_ATPase_subunit_A 1.725465725 2.31341E-38
1134109 - hypothetical_protein 1.742814111 1.03426E-19
1135580 serA D_3_phosphoglycerate_dehydrogenase 1.778877005 7.7274E-166
1134951 - sugar_ABC_transporter_permease 1.784809143 7.36705E-21
1135450 xylF xylose_ABC_transporter_substrate_binding_protein 1.790931796 2.1905E-133
1132539 acd acyl_CoA_dehydrogenase 1.793454494 2.3937E-168
1135755 - sugar_ABC_transporter_substrate_binding_protein 1.80599392 1.68203E-54
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1135113 - sugar_ABC_transporter_substrate_binding_protein 1.854646318 8.144E-109
1133847 - - 1.862515666 1.10224E-10
1135096 - ribose_ABC_transporter_substrate_binding_protein 1.873456903 1.495E-96
1136700 - ABC_transporter_substrate_binding_protein 1.87613774 2.1554E-167
1135128 - dicarboxylate_ABC_transporter_ATPase 1.877391279 1.2892E-113
1134569 actI actinorhodin_polyketide_dimerase 1.886500763 4.20958E-10
1132453 - hypothetical_protein 1.892021094 3.4481E-177
1135000 - ribose_ABC_transporter_substrate_binding_protein 1.925809343 1.3723E-156
1134939 - C4_dicarboxylate_binding_protein 1.944721126 5.17386E-57
1136061 - - 1.987796709 2.40425E-85
1136657 afuA ABC_transporter_substrate_binding_protein 2.009418548 1.6686E-105
1132443 fadD long_chain_fatty_acid_CoA_ligase 2.024272727 4.6578E-208
1132452 - - 2.02860773 2.73303E-51
1135349 - acetyl_CoA_C_acetyltransferase 2.047819195 1.124E-211
1132770 mmgC acyl_CoA_dehydrogenase 2.12685267 1.6647E-212
1135997 - branched_chain_amino_acid_ABC_transporter_substrate_binding_protein 2.155431593 2.5295E-120
1135242 - periplasmic_mannitol_binding_protein 2.297536557 8.1586E-247
1136322 - sorbitol_mannitol_ABC_transporter_permease 2.323099815 4.782E-208
1133047 - hypothetical_protein 2.420199014 3.1735E-245
1132143 - hypothetical_protein 2.423986428 1.09669E-05
1136321 - sorbitol_ABC_transporter_substrate_binding_protein 2.466994243 4.7163E-298
1134953 - sugar_ABC_transporter_substrate_binding_protein 2.513062188 4.5233E-168
1132156 - hypothetical_protein 2.533611732 1.6809E-259
1135127 - ABC_transporter_substrate_binding_protein 2.540687206 8.7498E-275
1134952 - sugar_ABC_transporter_permease 2.551030904 7.6627E-57
1140325 - hypothetical_protein 2.55624483 4.7361E-270
1133046 - hypothetical_protein 2.583562838 3.0512E-209
1135784 - LamB_YcsF_family_protein 2.585164923 2.0377E-287
1135912 - hypothetical_protein 2.699368077 0.000170523
1136693 - hypothetical_protein 2.886748538 1.1145E-246
1135785 - hypothetical_protein 3.137905687 3.2183E-299
1136395 - AraC_family_transcriptional_regulator 3.144633783 0
1132155 - hypothetical_protein 3.176404975 2.3779E-205
1135788 amyA alpha_amylase 3.213508065 2.574E-282
1135786 - hypothetical_protein 3.520265058 0
1136399 - oligopeptide_ABC_transporter_substrate_binding_protein 3.63903859 0
1136396 - hypothetical_protein 3.788970278 0
1132154 - hypothetical_protein 3.803902876 0
1136397 - oligopeptide_ABC_transporter_permease 3.814029577 0
1136398 - oligopeptide_ABC_transporter_permease 3.852438142 0
1135787 - biotin_carboxylase 3.883514872 0
1134530 mtbA MFS_permease 4.56809855 0
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![LSRB Project[1]](https://img.dokumen.tips/doc/110x75/543e7774afaf9f12698b4619/lsrb-project1.jpg)