Fur regulation on the capsular polysaccharide biosynthesis and iron-acquisition systems in Klebsiella pneumoniae CG43

Original Article

2

Fur regulation on the capsular polysaccharide biosynthesis and

iron-acquisition systems in Klebsiella pneumoniae CG43

5

Running title: Characterization of Fur in K. pneumoniae 6

Content category: Cell and Molecular Biology of Microbes 7

Ching-Ting Lin1*, Chien-Chen Wu2, Yu-Sheng Chen1, Yi-Chyi Lai3, Chia Chi1, 8

Jing-Ciao Lin1, Yeh Chen4, Hwei-Ling Peng2* 9

10

1

School of Chinese Medicine, China Medical University, Taichung, 40402, Taiwan.

11

Republic of China

12

2

Department of Biological Science and Technology, National Chiao Tung University,

13

Hsin Chu, 30068, Taiwan, Republic of China

14

3

Department of Microbiology and Immunology, Chung-Shan Medical University,

15

Taichung, 40201, Taiwan. Republic of China

16

4

Research Institute of Biotechnology, Hungkuang University, Taichung, 43302,

17

Taiwan, Republic of China

18 19 * Corresponding author. 20 21 Ching-Ting Lin 22

Postal address: School of Chinese Medicine, China Medical University, Taichung, 23

Taiwan, Republic of China. Phone: 886-4-22053366 ext. 56916. FAX: 24 886-4-5729288. E-mail: [email protected] 25 26 Hwei-Ling Peng 27

Postal address: Department of Biological Science and Technology, National Chiao 28

Tung University, Hsin Chu, 30068, Taiwan, Republic of China. Phone: 29

886-3-5727121 ext. 56916. FAX: 886-3-5729288. E-mail: [email protected] 30

31

Keywords: Fur, iron acquisition, capsular polysaccharide, Klebsiella pneumoniae 32

ABSTRACT

1 2

Ferric uptake regulator (Fur) has been reported to repress the expression of rmpA, a 3

regulatory gene for the mucoid phenotype, leading to the decrease of capsular 4

polysaccharide (CPS) biosynthesis in Klebsiella pneumoniae CG43. Here, 5

quantitative real-time polymerase chain reaction (qRT-PCR) analyses and 6

electrophoretic mobility shift assay showed that Fur also repressed the expression of 7

the CPS regulatory genes rmpA2 and rcsA. Interestingly, deletion of rmpA or rcsA but 8

not rmpA2 from the fur strain could suppress the deletion effect of Fur. The

9

availability of extracellular iron affected the CPS amount suggesting that Fur 10

regulates CPS biosynthesis in an Fe(II)-dependent manner. Increased production of 11

siderophores was observed in the fur strain suggesting the uptake of extracellular

12

iron in K. pneumoniae is regulated by Fur. Fur titration assay and qRT-PCR analyses 13

demonstrated that at least six of the eight putative iron-acquisition systems, identified 14

by a BLAST search in the contig database of K. pneumoniae CG43, were directly 15

repressed by Fur. Thus, we conclude that Fur has a dual role in the regulation of CPS 16

biosynthesis and iron acquisition in K. pneumoniae. 17

INTRODUCTION

1 2

Klebsiella pneumoniae is a rod-shaped Gram-negative bacterium that causes

3

community-acquired diseases including pneumonia, bacteremia, septicemia, and 4

urinary and respiratory tract infections, occurring particularly in 5

immune-compromised patients (Podschun & Ullmann, 1998). In Asian countries, 6

especially in Taiwan and Korea, K. pneumoniae is the predominant pathogen 7

responsible for pyogenic liver abscess in diabetic patients (Han, 1995; Lau et al., 2000; 8

Yang et al., 2009). Among the virulence factors identified in K. pneumoniae, capsular 9

polysaccharide (CPS) is considered as the major determinant for K. pneumoniae 10

infections. The pyogenic liver abscess isolates often carry heavy CPS that could 11

protect the bacteria from phagocytosis and killing by serum factors (Lin et al., 2004; 12

Sahly et al., 2000). Apart from the antiphagocytic function, Klebsiella CPS also helps 13

bacterial colonization and biofilm formation at the infection sites (Boddicker et al., 14

2006; Favre-Bonte et al., 1999; Moranta et al., 2010). 15

16

Rcs system is a well-known two-component system (2CS) that regulates the 17

expression of cps genes in bacteria (Stout, 1994). The transcription of cps genes is 18

controlled by the response regulator RcsB in complex with the auxiliary regulatory 19

protein RcsA. (Gottesman & Stout, 1991; Majdalani & Gottesman, 2005). Recently, 20

coordinated action of the 2CSs KvgAS, KvhAS, and KvhR, whereas gene regulation 1

is independent of RcsB. (Lin et al., 2006). Besides RcsA, the regulators RmpA and 2

RmpA2 also interact with RcsB for CPS biosynthesis regulation. Moreover, rmpA 3

expression was repressed by Fur, the global regulator for the expression of 4

iron-acquisition systems (Cheng et al., 2010). Whether Fur affects RcsA or RmpA2 is 5

yet to be investigated. 6

7

Under iron-repletion conditions, dimeric Fur in complex with Fe(II)binds to a 19-bp 8

consensus DNA sequence, the Fur box (GATAATGATwATCATTATC; w=A or T), in 9

the promoters of the genes required for iron uptake, thereby preventing transcription 10

from these genes (Griggs & Konisky, 1989). The regulation helps bacteria to avoid 11

iron overload, which may lead to the formation of hydroxyl radicals. Multiple 12

iron-acquisition systems are commonly present in bacteria for the uptake of iron in the 13

environment (Andrews et al., 2003). In an anaerobic environment, Fe(II) is prevalent 14

and is imported into the bacterial cytoplasm via the Feo system (Hantke, 2003). 15

However, in aerobic conditions and in mammalian tissues (in vivo), the majority of 16

iron is found as Fe(III), and iron in vivo is almost entirely sequestered by iron-binding 17

proteins (transferrin and lactoferrin) and hemoproteins (hemoglobin and myoglobin) 18

(Wandersman & Delepelaire, 2004). 19

1

Bacteria are generally equipped with iron/heme acquisition systems to directly 2

transport iron from the exogenous iron/heme sources or release siderophore and 3

hemophore compounds into the extracellular medium to scavenge iron/heme from 4

various sources (Wandersman & Delepelaire, 2004). In K. pneumoniae NTUH-K2044, 5

the expression of the ten putative iron-acquisition genes was highly up-regulated in 6

response to human serum, and bacterial virulence was decreased by the triple 7

mutation of siderophore genes (Hsieh et al., 2008). The siderophore genes 8

iucABCDiutA and iroNDCB also have been reported to be the determinants of K.

9

pneumoniae-caused liver abscess (KLA) (Hsieh et al., 2008; Koczura & Kaznowski,

10

2003; Tang et al., 2010). Nevertheless, the regulation of iron-acquisition gene 11

expression in K. pneumoniae has not yet been studied. 12

13

In this study, we investigated the regulatory roles of Fur on the expression of the cps 14

regulators RmpA, RmpA2, and RcsA, and the expression of eight iron-acquisition 15

systems in K. pneumoniae CG43. 16

MATERIAL AND METHODS

1 2

Bacterial strains, plasmids, and media. Bacterial strains and plasmids used in this

3

study are listed in Table 1. Bacteria were routinely cultured at 37°C in Luria-Bertani 4

(LB) medium or M9 minimal medium supplemented with appropriate antibiotics. The 5

antibiotics used include ampicillin (100 g/ml), kanamycin (25 g/ml), streptomycin 6

(500 g/ml), and tetracycline (12.5 g/ml). 7

Construction of deletion mutants. Specific gene deletions were introduced into K.

8

pneumoniae CG43 using an allelic exchange strategy as previously described (Lai et

9

al., 2003). The pKAS46 system was used in the selection of the mutants (Skorupski &

10

Taylor, 1996), and the mutations were confirmed by PCR and Southern hybridization 11

(data not shown). 12

Quantitative real-time polymerase chain reaction (qRT-PCR). Total RNAs were

13

isolated from bacteria cells grown to early exponential phase using the RNeasy 14

midi-column (QIAGEN) according to the manufacturer’s instructions. RNA was 15

treated with RNase-free DNase I (MoBioPlus) to eliminate DNA contamination. 16

Hundred nanogram of RNA was reverse-transcribed with the Transcriptor First Strand 17

cDNA Synthesis Kit (Roche) using random primers. qRT-PCR was performed in a 18

Roche LightCycler® 1.5 Instrument using LightCycler TaqMan Master (Roche). 19

Primers and probes were designed for selected target sequences using Universal 20

ProbeLibrary Assay Design Center (Roche-applied science) and are listed in Table 2. 1

Data were analyzed using the real time PCR software of Roche LightCycler® 1.5 2

Instrument. Relative gene expressions were quantified using the comparative 3

threshold cycle 2-CT method with 23S rRNA as the endogenous reference. 4

Electrophoretic mobility shift assay (EMSA)

5

Recombinant K. pneumoniae Fur protein was expressed in E. coli and purified as 6

previously described (Cheng et al., 2010). DNA fragments of the putative promoter 7

regions of rmpA, rmpA2, and rcsA were PCR amplified using specific primer sets. The 8

purified His6-Fur was incubated with 10-ng DNA in a 15-l solution containing 50

9

mM Tris-HCl (pH 7.5), 100 mM NaCl, 100 mM dithiothreitol, 200 M MnCl2, and 1

10

g/l BSA at room temperature for 20 min. The samples were then loaded onto 5% 11

native (nondenaturing) polyacrylamide gel containing 5% glycerol in 0.5 TB buffer 12

(45 mM Tris-HCl, pH 8.0, 45 mM boric acid) and electrophoresed at 20-mA constant 13

current at 4°C for 2 hr. The gel was stained with SYBR Green EMSA stain 14

(Invitrogen), and then visualized using the Safe Imager™ blue-light transilluminator. 15

Extraction and quantification of CPS. CPS was extracted and quantified as

16

previously described (Domenico et al., 1989). The glucuronic acid content, 17

representing the amount of K. pneumoniae K2 CPS, was determined from a standard 18

curve of glucuronic acid (Sigma-Aldrich) and expressed as micrograms per 109 CFU 19

(Blumenkrantz & Asboe-Hansen, 1973). 1

Identification of the iron acquisition genes in K. pneumoniae CG43. The ten genes

2

encoding different iron acquisition systems in K. pneumoniae NTUH-K2044 (Hsieh et 3

al., 2008) were used as query sequences to search for homologs in K. pneumoniae

4

CG43 contig database (unpublished results from Dr. S.-F. Tsai, National Health 5

Research Institutes, Taiwan) as assessed by the BLAST search program 6

(http://blast.ncbi.nlm.nih.gov/Blast.cgi) (Altschul et al., 1997). 7

Fur titration assay (FURTA). FURTA was performed according to the method

8

described by Stojiljkovic et al (Stojiljkovic et al., 1994). DNA sequences containing a 9

putative Fur box were PCR amplified with specific primer sets and then cloned into 10

pT7-7. The resulting plasmids were introduced into the E. coli strain H1717, and the 11

transformants were plated onto MacConkey-lactose plates containing 100 g/ml 12

ampicillin and 30 M Fe(NH4)2(SO4)2. The indicator strain H1717 contained a

13

chromosomal fhuF::lacZ fusion, and a low affinity Fur box has been demonstrated in 14

the fhuF promoter. The introduction of pT7-7 derived plasmids carrying Fur-binding 15

sequences could thus cause the removal of Fur from the fhuF Fur box (Hantke, 1987). 16

H1717 harboring pT7-7 was used as a negative control. Colony phenotype was 17

observed after incubation at 37°C for 10 h. Red colony (Lac+) denoted a 18

FURTA-positive phenotype and indicated the binding of Fur to the DNA sequence 19

cloned into the pT7-7 plasmid. 1

Chrome azurol S (CAS) assay. The CAS assay was performed according to the

2

method described by Schwyn and Neilands (Schwyn & Neilands, 1987). Each of the 3

bacterial strain was grown overnight in LB medium, and then 5 l of culture was 4

added onto a CAS agar plate. After 16 hr incubation at 37°C, effects of the bacterial 5

siderophore production could be observed. Siderophore production was apparent as an 6

orange halo around the colonies; absence of a halo indicated the inability to produce 7

siderophores. 8

Statistical method. Unpaired t test was used to determine the statistical significance

9

and values of P < 0.001 were considered significant. The results of CPS quantification 10

and qRT-PCR analysis were derived from a single experiment which is representative 11

of three independent experiments. Each sample was assayed in triplicate and the 12

average activity and standard deviation are presented. 13

14 15

RESULTS

1 2

Fur regulates the expression of RmpA, RmpA2, and RcsA 3

To investigate whether Fur affects the expression of the cps regulatory proteins 4

RcsA, RcsB, RmpA2, KvgA, and KvhR (Cheng et al., 2010; Lai et al., 2003; Lin et 5

al., 2006), in addition to RmpA (Cheng et al., 2010), qRT-PCR analyses were

6

performed to compare the expression levels in K. pneumoniae CG43S3 and its 7

isogenic fur strain. As shown in Fig. 1A, when the bacteria were grown in LB, the

8

deletion of fur increased the expression of not only rmpA but also rmpA2 and rcsA. 9

By contrast, fur deletion appeared to have no effect on the expression of rcsB, kvgA, 10

or kvhR. Inclusion of the iron chelator 2, 2-dipyridyl (Dip) in the growth medium 11

eliminated the effects caused by fur deletion, suggesting that a Fur-Fe(II) complex is 12

involved in regulating the expression of rmpA, rmpA2, and rcsA. Although the 13

expression of both rmpA and rcsA increased upon adding 200 M Dip, rmpA2 14

expression did not appear to change, suggesting a novel mechanism that requires 15

further study. 16

17

As in PrmpA, the promoter of rmpA, putative Fur box sequences could be found in the

18

upstream regions of rmpA2 and rcsA (Fig. 1B). We performed an EMSA to determine 19

whether Fur directly affects the expression of rmpA2 and rcsA. As shown in Fig. 1C, 20

the purified recombinant His6-Fur protein was able to bind to the upstream regions of

1

rmpA, rmpA2, and rcsA, but not to the P6 DNA which did not contain a Fur box

2

(Cheng et al., 2010). Addition of 200 M ethylenediaminetetraacetic acid (EDTA) to 3

the reaction mixture appeared to abolish the interactions (data not shown), indicating 4

that the formation of Fur-Fe(II) complex was required for the specific binding. 5

6

Fur repressed CPS biosynthesis via RmpA and RcsA 7

To investigate how Fur differentially regulates the expression of the three CPS 8

regulators, double mutants with a deletion of rmpA, rmpA2, or rcsA from the fur

9

strain background were constructed, and the effects of the mutations on bacterial CPS 10

biosynthesis were assessed. Consistent with previous reports (Cheng et al., 2010; Ebel 11

& Trempy, 1999; Lai et al., 2003), deletion of rmpA, rmpA2, or rcsA caused a 12

reduction in the amount of bacterial CPS (Fig. 2). By contrast, a significant increase in 13

CPS amount was found in the fur strain. Interestingly, deletion of rmpA or rcsA, but

14

not rmpA2, suppressed the fur deletion phenotype (Fig. 2). The results suggest that the 15

activation of CPS biosynthesis in the fur strain is mediated by RmpA or RcsA, but

16

not RmpA2, under the assay conditions. 17

18

It has been reported that the K2 cps gene cluster of K. pneumoniae Chedid contains 19 19

open reading frames (ORFs) organized into three transcription units, orf1-2, orf3-15, 1

and orf16-17 (Arakawa et al., 1995). Analysis of the cps promoters revealed no 2

conserved Fur box, suggesting that Fur exerts indirect control over the transcription of 3

cps. To investigate this possibility, transcripts of orf1, orf3, and orf16 in wild-type

4

(CG43S3), fur,rmpA, rmpA2, rcsA, furrmpA, furrmpA2, furrcsA,

5

furrmpArcsA, and furrmpArmpA2rcsA strains were measured using

6

qRT-PCR. As shown in Fig. 3A–C, all three transcripts were differentially decreased 7

in rmpA, rmpA2, and rcsA strains. Compared to either the rmpA or rcsA deletions,

8

the deletion of rmpA2 had less effect on the transcription of orf1, orf3, and orf16. 9

Interestingly, rmpA deletion had more profound reducing effects on the transcription 10

of orf1 and orf16 than rcsA deletion. Moreover, the cps expression levels in rmpA,

11

rmpArcsA, and rmpArmpA2rcsA were similar, suggesting a major regulatory

12

role of RmpA for controlling cps expression. However, RcsA and RmpA2 may also 13

play a major role in cps expression under conditions that have not been identified. 14

Moreover, further study is needed to determine whether a regulatory interaction exists 15

between RmpA, RmpA2, and RcsA. 16

17

Consistent with the results shown in Fig. 2, the deletion effect of fur was eliminated in 18

the furrmpA or furrcsA strains when the orf1 and orf16 transcripts were

expressed (Fig. 3A and C). Deletion of rmpA from the fur strain significantly

1

decreased the level of all three cps transcripts. The quantities of the cps transcripts in 2

furrmpArcsA or furrmpArmpA2rcsA were similar to that of the furrmpA

3

strain. These results further support the assumption that RmpA plays a major role in 4

the Fur-mediated repression of cps transcription. By contrast, no apparent difference 5

in cps expression was observed between fur and furrmpA2, indicating that a 6

minor role, if any, in the Fur-mediated regulation of cps expression. Nevertheless, the 7

much higher expression levels of cps that were observed in furrmpArmpA2rcsA

8

than the strain rmpArmpA2rcsA suggest that an unknown regulator may be

9

involved in the Fur-mediated control of cps expression. 10

11

Availability of iron affects CPS biosynthesis in K. pneumoniae 12

To determine whether Fur regulates gene expression in an Fe(II)-dependent manner 13

(Andrews et al., 2003; Escolar et al., 1999), we analyzed the effects of iron depletion 14

and iron repletion on CPS biosynthesis. As shown in Fig. 4, the CPS amount was 15

increased in the fur strain when the bacteria were grown in LB medium containing

16

~18 M iron (Abdul-Tehrani et al., 1999). The fur deletion effect was no longer 17

observed in the fur-complement strain, nor was it observed when Dip was added to 18

the growth medium. In addition, the addition of 60 M FeSO4 in M9 medium caused

an apparent decrease in the amount of CPS in the wild-type strain compared to that of 1

wild-type strain grown only in M9 medium. The fur strain grown in M9 medium

2

both with and without FeSO4 produced a higher amount of CPS than the wild-type

3

strain, indicating that an iron level of approximately 2 M in M9 medium 4

(Abdul-Tehrani et al., 1999) may be sufficient for Fur activity to repress CPS 5

biosynthesis. These results suggest that iron repletion increased Fur activity, thereby 6

repressing the biosynthesis of CPS. 7

8

The regulatory role of Fur in iron-acquisition systems of K. pneumoniae CG43 9

To assess whether Fur affects iron-acquisition in K. pneumoniae as in other bacteria, a 10

CAS assay was performed to analyze the activity of siderophore secreted. As shown 11

in Fig. 5A, an orange halo around the colony of K. pneumoniae fur strain grown on a

12

blue CAS plate was observed. Introduction of the complement plasmid pfur into the 13

fur strain appeared to diminish the orange halo phenotype. A BLAST search using

14

the DNA sequences of the iron-acquisition systems in K. pneumoniae NTUH-K2044 15

as templates (Hsieh et al., 2008) for the homologs in the contig database of K. 16

pneumoniae CG43 (unpublished results from Dr. S.-F. Tsai, National Health Research

17

Institutes, Taiwan) was subsequently performed. As shown in Table 3, eight putative 18

iron-acquisition systems were identified. Expression of the genes (iucA, fepA, fepB, 19

entC, iroB, hmuR, and feoB), corresponding to five iron-acquisition systems assessed

1

using qRT-PCR, were increased at least two-fold in fur strain. Expression of fhuA,

2

fecA, fecE, and sitA genes was also activated in fur strain, although with less than

3

two-fold increase (Table 3). 4

5

As shown in Fig. 5B, homologous sequences of the Fur box (de Lorenzo et al., 6

1987) could be identified in the putative promoters PiroB, PentC, PhmuR, Pfeo, Pfec, Pfhu and

7

Psit. A Fur box homolog was also found in the coding region of iucA, at the position -4

8

to +15 relative to the start codon. These Fur box-containing DNA fragments were then 9

cloned into pT7-7, and the resulting plasmids were introduced individually into the E. 10

coli indicator strain H1717. As shown in Fig. 5C, the E. coli H1717 harboring the

11

plasmid with PiucA, PiroB, PentC, PhmuR, Pfeo, or Pfec, showed FURTA-positive phenotypes.

12

While the H1717 strains harboring pT7-7 derivatives with the upstream regions of 13

fhuA or sitA exhibited a FURTA-negative phenotype. The results suggest that Fur can

14

bind to each of the predicted Fur box sequences on iroB, entC, iucA, hmuR, feoB, and 15

fecA to exert its regulatory function in vivo.

16 17

Extracellular Fe(II) has been demonstrated to be transported into bacteria via the iron 18

acquisition systems FeoABC and SitABCD (Cartron et al., 2006; Sabri et al., 2006). 19

As shown in Fig. 5, expression of the feo but not the sit genes was affected by Fur. 1

The feoB deletion mutant, which was predicted to decrease the bacterial 2

Fe(II)-transport ability, was therefore generated to investigate if the Fe(II)-dependent 3

regulation of CPS biosynthesis is affected by the Feo system. However, no difference 4

in CPS amount between the wild-type and feoB strains, grown in both LB and M9

5

supplemented with various concentrations of Dip or FeSO4, was found (data not

6

shown). It is possible that the SitABCD or other iron acquisition systems are involved 7

in the Fur-Fe(II)-dependent regulation on CPS biosynthesis, which may then 8

compensate the mutation effect of feoB. 9

DISCUSSION

1 2

In this study, we demonstrated that Fur direct controls the expression of the CPS 3

regulators RmpA, RmpA2, and RcsA (Fig. 1). It has been reported previously that fur 4

mutation does not produce an obvious change in rmpA2 promoter activity, as assessed 5

by the lacZ reporter system (Cheng et al., 2010). By contrast, qRT-PCR analysis 6

revealed that deletion of fur caused an approximately two-fold increase in rmpA2 7

mRNA (Fig. 1A). The discrepancy may be due to the dosage effect of the 8

plasmid-based lacZ reporter system, which is known to over-estimate -galactosidase 9

activity. The EMSA results shown in Fig. 1C also support the direct binding of Fur to 10

the rmpA2 promoter. Because the rmpA2 promoter does not fit well with the Fur of E. 11

coli, it remains to be investigated whether K. pneumoniae Fur exerts less rigid

12

recognition sequences. 13

14

The two homologous genes rmpA and rmpA2 are on pLVPK, and both encode CPS 15

regulators for the activation of CPS biosynthesis (Chen et al., 2004; Lai et al., 2003). 16

Compared to RmpA, RmpA2 has an extended N-terminal region and a different 17

promoter sequence, which implied that the two transcriptional factors are functionally 18

different. As shown in Fig. 2, the deleting effect of fur was eliminated by the further 19

roles in the regulation of CPS biosynthesis. Further investigation is needed to clarify 1

the roles of the two homologous regulators in K. pneumoniae. 2

3

Fur has been demonstrated to be a global regulator in many bacteria (Cornelis et al., 4

2009; Mey et al., 2005; Moore & Helmann, 2005). Recently, the deletion of fur in 5

Helicobacter pylori was shown to reduce the expression of Lon protease (Choi et al.,

6

2009), which can affect the protein stability of RcsA and RmpA2 in E. coli and K. 7

pneumoniae (Lai et al., 2003; Trisler & Gottesman, 1984). However, fur deletion in K.

8

pneumoniae CG43 reveals no obvious effect on the expression of lon (data not shown).

9

The Fur protein sequences of H. pylori and K. pneumoniae have low identity (25.6%), 10

suggesting that the Fur regulatory circuit is different in the two bacteria. 11

12

The K2 cps gene cluster is predicted to encode proteins that are involved in the 13

synthesis, transport, assembly, and modification of CPS (Whitfield & Roberts, 1999). 14

As shown in Fig. 3, the differential regulations exerted by RmpA, RmpA2, and RcsA 15

on cps expression affect both the amount and composition of CPS. Further studies 16

should investigate whether RmpA, RmpA2, and RcsA also affect CPS modifications, 17

thus influencing the interactions between bacteria and host cells. The mutant 18

furrmpArmpA2rcsA had a higher level of cps expression than the mutant

rmpArmpA2rcsA, indicating that one or more unknown regulators besides RmpA,

1

RmpA2, and RcsA may be involved in the Fur-mediated control of cps transcription. 2

The complex regulation of cps expression in K. pneumoniae requires further 3

exploration. 4

5

In K. pneumoniae, Fur regulates the expression of flavodoxin and CPS biosynthesis, 6

in addition to regulating its own expression (Achenbach & Genova, 1997; Achenbach 7

& Yang, 1997; Cheng et al., 2010). Here, we showed that Fur serves as a repressor in 8

the regulation of at least eight iron-acquisition systems in K. pneumoniae CG43, 9

although at different levels (Table 3). Analysis of the putative Fur boxes on iroB, entC, 10

hmuR, iucA, feo, and fec revealed high identities to the consensus sequence (15-16 of

11

19 positions), whereas those of fhuA and sitA exhibited relatively lower identities (13 12

of 19 positions). This suggests that a highly conserved sequence of the nineteen base 13

pairs sequence is required for a positive FURTA phenotype. During infection, 14

differential expression of the iron-acquisition system is anticipated to provide an 15

adaptive advantage because of its flexibility in responding to various environmental 16

stimuli (Caza et al., 2008; Valdebenito et al., 2006). Therefore, it is predicated that the 17

eight iron-acquisition systems in CG43 are coordinated differently. Whether CG43 18

harbors other iron-acquisition genes remains to be further investigated. 19

In this study, we characterized the role of Fur in the CPS regulatory circuit of K. 1

pneumoniae CG43, and found that RmpA, RcsA, and RmpA2 are directly regulated

2

by Fur. We also demonstrated that Fur regulates CPS biosynthesis via RcsA or RmpA, 3

but not RmpA2, in an Fe(II)-dependent manner. Moreover, we report a fur deletion 4

effect on the expression of the eight iron-acquisition systems identified in K. 5

pneumoniae CG43.

6 7

1

ACKNOWLEDGEMENTS

2 3

We are grateful to Dr. Hantke for providing the E. coli strain H1717. The work is 4

supported by the grants from National Science Council (NSC 5

97-2314-B-039-042-MY2) and China Medical University (CMU97-204 and 6

CMU97-345) to CT Lin, and NSC 97-2320-B-009-001-MY3 to HL Peng. 7

REFERENCE

1 2 3

Abdul-Tehrani, H., Hudson, A. J., Chang, Y. S., Timms, A. R., Hawkins, C.,

4

Williams, J. M., Harrison, P. M., Guest, J. R. & Andrews, S. C. (1999). Ferritin

5

mutants of Escherichia coli are iron deficient and growth impaired, and fur mutants 6

are iron deficient. J Bacteriol 181, 1415-1428. 7

8

Achenbach, L. A. & Genova, E. G. (1997). Transcriptional regulation of a second

9

flavodoxin gene from Klebsiella pneumoniae. Gene 194, 235-240. 10

11

Achenbach, L. A. & Yang, W. (1997). The fur gene from Klebsiella pneumoniae:

12

characterization, genomic organization and phylogenetic analysis. Gene 185, 201-207. 13

14

Altschul, S. F., Madden, T. L., Schaffer, A. A., Zhang, J., Zhang, Z., Miller, W. &

15

Lipman, D. J. (1997). Gapped BLAST and PSI-BLAST: a new generation of protein

16

database search programs. Nucleic Acids Res 25, 3389-3402. 17

18

Andrews, S. C., Robinson, A. K. & Rodriguez-Quinones, F. (2003). Bacterial iron

19

homeostasis. FEMS Microbiol Rev 27, 215-237. 20

21

Arakawa, Y., Wacharotayankun, R., Nagatsuka, T., Ito, H., Kato, N. & Ohta, M.

22

(1995). Genomic organization of the Klebsiella pneumoniae cps region responsible

23

for serotype K2 capsular polysaccharide synthesis in the virulent strain Chedid. J 24

Bacteriol 177, 1788-1796.

25 26

Blumenkrantz, N. & Asboe-Hansen, G. (1973). New method for quantitative

27

determination of uronic acids. Anal Biochem 54, 484-489. 28

29

Boddicker, J. D., Anderson, R. A., Jagnow, J. & Clegg, S. (2006). Signature-tagged

30

mutagenesis of Klebsiella pneumoniae to identify genes that influence biofilm 31

formation on extracellular matrix material. Infect Immun 74, 4590-4597. 32

33

Braun, V. & Mahren, S. (2005). Transmembrane transcriptional control (surface

34

signalling) of the Escherichia coli Fec type. FEMS Microbiol Rev 29, 673-684. 35

36

Cartron, M. L., Maddocks, S., Gillingham, P., Craven, C. J. & Andrews, S. C.

(2006). Feo--transport of ferrous iron into bacteria. Biometals 19, 143-157.

1 2

Caza, M., Lepine, F., Milot, S. & Dozois, C. M. (2008). Specific roles of the

3

iroBCDEN genes in virulence of an avian pathogenic Escherichia coli O78 strain and

4

in production of salmochelins. Infect Immun 76, 3539-3549. 5

6

Chen, Y. T., Chang, H. Y., Lai, Y. C., Pan, C. C., Tsai, S. F. & Peng, H. L. (2004).

7

Sequencing and analysis of the large virulence plasmid pLVPK of Klebsiella 8

pneumoniae CG43. Gene 337, 189-198.

9 10

Cheng, H. Y., Chen, Y. S., Wu, C. Y., Chang, H. Y., Lai, Y. C. & Peng, H. L.

11

(2010). RmpA regulation of capsular polysaccharide biosynthesis in Klebsiella

12

pneumoniae CG43. J Bacteriol 192, 3144-3158.

13 14

Choi, Y. W., Park, S. A., Lee, H. W. & Lee, N. G. (2009). Alteration of

15

growth-phase-dependent protein regulation by a fur mutation in Helicobacter pylori. 16

FEMS Microbiol Lett 294, 102-110.

17 18

Cornelis, P., Matthijs, S. & Van Oeffelen, L. (2009). Iron uptake regulation in

19

Pseudomonas aeruginosa. Biometals 22, 15-22.

20 21

de Lorenzo, V., Wee, S., Herrero, M. & Neilands, J. B. (1987). Operator sequences

22

of the aerobactin operon of plasmid ColV-K30 binding the ferric uptake regulation 23

(fur) repressor. J Bacteriol 169, 2624-2630. 24

25

Domenico, P., Schwartz, S. & Cunha, B. A. (1989). Reduction of capsular

26

polysaccharide production in Klebsiella pneumoniae by sodium salicylate. Infect 27

Immun 57, 3778-3782.

28 29

Ebel, W. & Trempy, J. E. (1999). Escherichia coli RcsA, a positive activator of

30

colanic acid capsular polysaccharide synthesis, functions To activate its own 31

expression. J Bacteriol 181, 577-584. 32

33

Escolar, L., Perez-Martin, J. & de Lorenzo, V. (1999). Opening the iron box:

34

transcriptional metalloregulation by the Fur protein. J Bacteriol 181, 6223-6229. 35

36

Favre-Bonte, S., Joly, B. & Forestier, C. (1999). Consequences of reduction of

37

Klebsiella pneumoniae capsule expression on interactions of this bacterium with

epithelial cells. Infect Immun 67, 554-561. 1

2

Ferguson, A. D., Hofmann, E., Coulton, J. W., Diederichs, K. & Welte, W. (1998).

3

Siderophore-mediated iron transport: crystal structure of FhuA with bound 4

lipopolysaccharide. Science 282, 2215-2220. 5

6

Gottesman, S. & Stout, V. (1991). Regulation of capsular polysaccharide synthesis in

7

Escherichia coli K12. Mol Microbiol 5, 1599-1606.

8 9

Griggs, D. W. & Konisky, J. (1989). Mechanism for iron-regulated transcription of

10

the Escherichia coli cir gene: metal-dependent binding of fur protein to the promoters. 11

J Bacteriol 171, 1048-1054.

12 13

Han, S. H. (1995). Review of hepatic abscess from Klebsiella pneumoniae. An

14

association with diabetes mellitus and septic endophthalmitis. West J Med 162, 15

220-224. 16

17

Hanahan, D. (1983). Studies on transformation of Escherichia coli with plasmids. J

18

Mol Biol 166, 557-580.

19 20

Hantke, K. (1987). Selection procedure for deregulated iron transport mutants (fur) in

21

Escherichia coli K 12: fur not only affects iron metabolism. Mol Gen Genet 210,

22

135-139. 23

24

Hantke, K. (2003). Is the bacterial ferrous iron transporter FeoB a living fossil?

25

Trends Microbiol 11, 192-195.

26 27

Hsieh, P. F., Lin, T. L., Lee, C. Z., Tsai, S. F. & Wang, J. T. (2008). Serum-induced

28

iron-acquisition systems and TonB contribute to virulence in Klebsiella pneumoniae 29

causing primary pyogenic liver abscess. J Infect Dis 197, 1717-1727. 30

31

Keen, N. T., Tamaki, S., Kobayashi, D. & Trollinger, D. (1988). Improved

32

broad-host-range plasmids for DNA cloning in gram-negative bacteria. Gene 70, 33

191-197. 34

35

Koczura, R. & Kaznowski, A. (2003). Occurrence of the Yersinia high-pathogenicity

36

island and iron uptake systems in clinical isolates of Klebsiella pneumoniae. Microb 37

Pathog 35, 197-202.

1

Lai, Y. C., Peng, H. L. & Chang, H. Y. (2001). Identification of genes induced in

2

vivo during Klebsiella pneumoniae CG43 infection. Infect Immun 69, 7140-7145. 3

4

Lai, Y. C., Peng, H. L. & Chang, H. Y. (2003). RmpA2, an activator of capsule

5

biosynthesis in Klebsiella pneumoniae CG43, regulates K2 cps gene expression at the 6

transcriptional level. J Bacteriol 185, 788-800. 7

8

Lau, Y. J., Hu, B. S., Wu, W. L., Lin, Y. H., Chang, H. Y. & Shi, Z. Y. (2000).

9

Identification of a major cluster of Klebsiella pneumoniae isolates from patients with 10

liver abscess in Taiwan. J Clin Microbiol 38, 412-414. 11

12

Lin, C. T., Huang, T. Y., Liang, W. C. & Peng, H. L. (2006). Homologous response

13

regulators KvgA, KvhA and KvhR regulate the synthesis of capsular polysaccharide 14

in Klebsiella pneumoniae CG43 in a coordinated manner. J Biochem (Tokyo) 140, 15

429-438. 16

17

Lin, J. C., Chang, F. Y., Fung, C. P., Xu, J. Z., Cheng, H. P., Wang, J. J., Huang,

18

L. Y. & Siu, L. K. (2004). High prevalence of phagocytic-resistant capsular serotypes

19

of Klebsiella pneumoniae in liver abscess. Microbes Infect 6, 1191-1198. 20

21

Majdalani, N. & Gottesman, S. (2005). The Rcs Phosphorelay: A Complex Signal

22

Transduction System. Annu Rev Microbiol. 23

24

Mey, A. R., Wyckoff, E. E., Kanukurthy, V., Fisher, C. R. & Payne, S. M. (2005).

25

Iron and fur regulation in Vibrio cholerae and the role of fur in virulence. Infect 26

Immun 73, 8167-8178.

27 28

Moore, C. M. & Helmann, J. D. (2005). Metal ion homeostasis in Bacillus subtilis.

29

Curr Opin Microbiol 8, 188-195.

30 31

Moranta, D., Regueiro, V., March, C., Llobet, E., Margareto, J., Larrate, E.,

32

Garmendia, J. & Bengoechea, J. A. (2010). Klebsiella pneumoniae capsule

33

polysaccharide impedes the expression of beta-defensins by airway epithelial cells. 34

Infect Immun 78, 1135-1146.

35 36

Nassif, X. & Sansonetti, P. J. (1986). Correlation of the virulence of Klebsiella

37

pneumoniae K1 and K2 with the presence of a plasmid encoding aerobactin. Infect

Immun 54, 603-608.

1 2

Podschun, R. & Ullmann, U. (1998). Klebsiella spp. as nosocomial pathogens:

3

epidemiology, taxonomy, typing methods, and pathogenicity factors. Clin Microbiol 4

Rev 11, 589-603.

5 6

Sabri, M., Leveille, S. & Dozois, C. M. (2006). A SitABCD homologue from an

7

avian pathogenic Escherichia coli strain mediates transport of iron and manganese 8

and resistance to hydrogen peroxide. Microbiology 152, 745-758. 9

10

Sahly, H., Podschun, R., Oelschlaeger, T. A. & other authors (2000). Capsule

11

impedes adhesion to and invasion of epithelial cells by Klebsiella pneumoniae. Infect 12

Immun 68, 6744-6749.

13 14

Schwyn, B. & Neilands, J. B. (1987). Universal chemical assay for the detection and

15

determination of siderophores. Anal Biochem 160, 47-56. 16

17

Skorupski, K. & Taylor, R. K. (1996). Positive selection vectors for allelic exchange.

18

Gene 169, 47-52.

19 20

Stojiljkovic, I., Baumler, A. J. & Hantke, K. (1994). Fur regulon in gram-negative

21

bacteria. Identification and characterization of new iron-regulated Escherichia coli 22

genes by a fur titration assay. J Mol Biol 236, 531-545. 23

24

Stout, V. (1994). Regulation of capsule synthesis includes interactions of the

25

RcsC/RcsB regulatory pair. Res Microbiol 145, 389-392. 26

27

Tabor, S. & Richardson, C. C. (1985). A bacteriophage T7 RNA

28

polymerase/promoter system for controlled exclusive expression of specific genes. 29

Proc Natl Acad Sci U S A 82, 1074-1078.

30 31

Tang, H. L., Chiang, M. K., Liou, W. J. & other authors (2010). Correlation

32

between Klebsiella pneumoniae carrying pLVPK-derived loci and abscess formation. 33

Eur J Clin Microbiol Infect Dis 29, 689-698.

34 35

Thompson, J. M., Jones, H. A. & Perry, R. D. (1999). Molecular characterization of

36

the hemin uptake locus (hmu) from Yersinia pestis and analysis of hmu mutants for 37

hemin and hemoprotein utilization. Infect Immun 67, 3879-3892. 38

1

Trisler, P. & Gottesman, S. (1984). lon transcriptional regulation of genes necessary

2

for capsular polysaccharide synthesis in Escherichia coli K-12. J Bacteriol 160, 3

184-191. 4

5

Valdebenito, M., Crumbliss, A. L., Winkelmann, G. & Hantke, K. (2006).

6

Environmental factors influence the production of enterobactin, salmochelin, 7

aerobactin, and yersiniabactin in Escherichia coli strain Nissle 1917. Int J Med 8

Microbiol 296, 513-520.

9 10

Wandersman, C. & Delepelaire, P. (2004). Bacterial iron sources: from siderophores

11

to hemophores. Annu Rev Microbiol 58, 611-647. 12

13

Whitfield, C. & Roberts, I. S. (1999). Structure, assembly and regulation of

14

expression of capsules in Escherichia coli. Mol Microbiol 31, 1307-1319. 15

16

Yang, Y. S., Siu, L. K., Yeh, K. M., Fung, C. P., Huang, S. J., Hung, H. C., Lin, J.

17

C. & Chang, F. Y. (2009). Recurrent Klebsiella pneumoniae liver abscess: clinical

18

and microbiological characteristics. J Clin Microbiol 47, 3336-3339. 19

20 21 22 23

FIGURE LEGENDS

1

Figure 1. Fur directly repressed the expression of rmpA, rmpA2, and rcsA. (A)

2

qRT-PCR analysis. The K. pneumoniae CG43S3 [pRK415], fur [pRK415], and fur

3

[pfur] strains were grown overnight in LB both with and without 200 M 2, 4

2-dipyridyl (Dip), then the relative expression of rmpA, rmpA2, rcsA, rcsB, kvgA, and 5

kvhR in bacteria were measured by qRT-PCR analysis. (B) DNA sequence alignment

6

between the E. coli typical Fur box and the putative Fur boxes in the upstream regions 7

of rmpA, rmpA2, and rcsA. The relative positions to the translational start sites are 8

indicated. (C) EMSA of the recombinant His6-Fur and its target promoters. DNAs of

9

the upstream regions of rmpA, rmpA2, and rcsA were incubated with an increasing 10

amount of the His6-Fur for 30 min and then loaded onto a 5% non-denaturing

11

polyacrylamide gel. The DNA fragment P6 was used as a negative control. The gel 12

was stained with SYBR Green EMSA stain and imaged. 13

Figure 2. Fur represses CPS biosynthesis via RmpA and RcsA. Bacteria strains, as

14

indicated in the margin, were grown in LB medium at 37℃ with agitation. After 16 hr 15

growth, the bacterial glucuronic acid contents were determined. Values are mean ± 16

standard error of three independent experiments. 17

Figure 3. qRT-PCR analyses of the expression of the K2 cps genes. Bacteria strains,

18

as indicated in the margin, were grown in LB medium at 37℃ with agitation and then 19

subjected to qRT-PCR analyses for the detection of orf1 (A), orf3 (B), and orf16 (C) 20

expression. 21

Figure 4. Fur affects the K. pneumoniae CPS biosynthesis in a Fe(II)-dependent

22

manner. Bacteria were grown in media supplemented both with and without either

23

200 M Dip or 60 M FeSO4 as indicated. After 16 hr growth, the bacterial

24

glucuronic acid contents were determined. Values are mean ± standard error of three 25

independent experiments. 26

Figure 5. Fur regulation on iron acquisition in K. pneumoniae CG43. (A) Deletion

27

of fur increases the K. pneumoniae siderophore production assessed using CAS assay. 28

Each bacterial strain assayed is indicated, and the orange halos formed around the 29

colonies correspond to iron-chelating activity of siderophore in bacteria. (B) DNA 30

sequence alignment between the E. coli typical Fur box and the putative Fur boxes in 31

the upstream regions of the eight iron acquisition systems. Positions identical to the 32

consensus sequences are underlined. (C) Assessment of the binding of Fur to the 33

DNA sequences using FURTA. E. coli H1717 strains carrying the pT7-7 derivatives 34

are indicated. Red colonies (Lac+) denoted FURTA-positive phenotypes. pT7-7, the 35

Table 1. Bacterial strains and plasmids used in this study

1 2

Strains or plasmids Descriptions Reference or source

K. pneumoniae

CG43S3 CG43 Smr _{(Lai et al., 2001)}

rmpA CG43S3rmpA (Cheng et al., 2010)

rmpA2 CG43S3rmpA2 (Lai et al., 2001)

fur CG43S3fur (Cheng et al., 2010)

rcsA CG43S3rcsA This study

rmpArcsA CG43S3rmpArcsA This study

rmpArmpA2rcsA CG43S3rmpArmpA2rcsA This study

furrmpA CG43S3furrmpA This study

furrmpA2 CG43S3furrmpA2 This study

furrcsA CG43S3furrcsA This study

furrmpArcsA CG43S3furrmpArcsA This study

furrmpArmpA2rcsA CG43S3furrmpArmpA2rcsA This study

E. coli

DH5 supE44 lacU169 (f80 lacZhsdRrecA1 endA1 gyrA96 thi-1 relA1

(Hanahan, 1983) BL21-RIL F- ompT hsdSB[rB-mB-]gal dcm [DE3] Laboratory stock

S17-1  pir hsdR recA pro RP4-2 [Tc::Mu; Km::Tn7] [pir (Skorupski & Taylor, 1996) H1717 araD139 lacU169 rpsL150 relA1 flbB5301 deoC1 ptsF25

rbsR aroB fhuF:: placMu

(Hantke, 1987) Plasmids

pKAS46 Positive selection suicide vector, rpsL Apr Kmr (Skorupski & Taylor, 1996) pET30a-c His-tagging protein expression vector, Kmr _Novagen

yT&A TA cloning vector Yeastern

pRK415 Broad-host-range IncP cloning vector, Tcr _{(Keen et al., 1988)}

pT7-7 Cloning vector, Apr _{(Tabor & Richardson, 1985)}

pfur03 1.7 kb fragment containing an internal 454 bp deletion in fur cloned into pKAS46

(Cheng et al., 2010) prcsA03 2.0 kb fragment containing an internal 620 bp deletion in

rcsA cloned into pKAS46

This study piroB_2 928 bp fragment containing the putative iroBCD promoter,

cloned into pT7-7

This study pentC_2 284 bp fragment containing the putative entC promoter,

cloned into pT7-7

This study piucA_2 700 bp fragment containing the putative iucABCD promoter,

cloned into pT7-7

This study phmuR_2 500 bp fragment containing the putative hmuRSTUV

promoter, cloned into pT7-7

This study pfeo_2 564 bp fragment containing the putative feoABC promoter,

cloned into pT7-7

This study pfec_2 296 bp fragment containing the putative fecIRA promoter,

cloned into pT7-7

This study pfhuA_2 313 bp fragment containing the putative fhuA promoter,

cloned into pT7-7

This study psitA_2 283 bp fragment containing the putative sitABCD promoter,

cloned into pT7-7

This study pFT01 0.5 kb fragment containing the putative orf1-2 promoter,

cloned into pT7-7

This study pFT02 0.9 kb fragment containing the putative orf3-15 promoter,

cloned into pT7-7

This study pFT03 0.3 kb fragment containing the putative orf16-17 promoter,

cloned into pT7-7

This study pFT04 0.5 kb fragment containing the putative rmpA promoter,

cloned into pT7-7

cloned into pT7-7

pFT06 0.5 kb fragment containing the putative rcsA promoter, cloned into pT7-7

This study

Table 2. Primers used in this study

1 2

Primer Sequence (5’3’) Enzyme cleaved Target

For FURTA

FA01 GAAGCTTGGAGCGCAGTTAGCGGAC HindIII

PiroB

FA02 CGGATCCGCCCATAGAGAGGAGGACC BamHI

FA03 GAAGCTTCCTGGGCTGAGGTAATTCC HindIII

PentC

FA04 CGGATCCCTCAGCCAGTGACGTTTCC BamHI

FA05 GGATCCAGAGGGTGATTTGCCAGCAT BamHI

PiucA

FA06 AGATCTGGAAGCACTGAGCAGCCACA BglII

FA07 ACACCAAGCTTCTGACGGAG HindIII

PhmuR

FA08 CTCCGGGATCCAGACATCGC BamHI

FA09 GGATCCCAACAGCGCGATGATGGAT BamHI

Pfeo

FA10 AGATCTGCCAGCATGCCGAGGGAGA BglII

FA11 GAAGCTTGTCGCGGGCTGGATCAAG HindIII

PfhuA

FA12 CGGATCCCGCAGCGAGTGATTTGGC BamHI

FA13 GAATTCGCAGCCTGATTGAC EcoRI

PsitA

FA14 GGTGTAGCATAGGATCCCTC BamHI

For qRT-PCR Sequence (5’3’) TaqMan probes Target

GT56 ACCCCGCCAGCTTTAACTT 3 entC GT57 TGTCCTTCTTTACGCAGCAG GT58 CAACCTGAACAGCGATTTCC 20 fecA GT59 TCGGCGCTCTCTTTAACAGT GT62 CAGATGTCAGCGCAGATCC 20 feoB GT63 CATAGGCCCGGCTGTAGA GT64 AAAGAGATTGGCCTCGAGTTT 20 fepA GT65 TGTTGCGGTAGTCGTTGC GT66 AATAAACAGCTCGTTTCGTTAAAAG 160 fepB GT67 GTATAGACCAGGGCGGTCAC GT68 GTTTGGTCGTATCGCCTGAC 3 fhuA GT69 GGAAGGTGAAGTCAGTTTTATCG GT72 TGATGACCTACCTGCAGTACCA 20 hmuR GT73 GAGCCGAGGTTCCAGGAG GT74 CGGAGGAACATTCGTCAAA 84 iroB GT75 TTCGGAATCTAAGCCTGGTG GT78 TCTCCCGGCTTATTGTTGATA 67 iucA GT79 GGAAGGTTTCGCAACTGGT GT82 GAAGATCCGTCAGACGATGG 20 sitA GT83 TAGTCGCGGGCCAGATAG RT03 CGTCATCCAGACCAAAGAGC 83 orf1 RT04 CCGGTTTTTCAATAAACTCGAC RT05 CGATGACCGGCTTTTTAATG 83 orf3 RT06 CTAGCGGAGATTTGGTACTGC RT07 CAGTCCACCTTTATTCCGATTG 67 orf16 RT08 AGGTACGACCCCGACTGG RT11 GGTAGGGGAGCGTTCTGTAA 67 23S rRNA RT12 TCAGCATTCGCACTTCTGAT RT17 TCAATAGCAATTAAGCACAAAAGAA 18 rmpA RT18 TTGTACCCTCCCCATTTCC RT19 AAATCATTACCCACAACTAACAAAAA 80 rmpA2 RT20 TTAGACGGCTTTTTAATTCATGG GT25 AAAACAGAATCAAATATGCTGCAA 158 rcsA GT26 CGTTGAGATTTGCGAAGTACC RT31 AAATTCACCCCGGAAAGC 120 rcsB RT32 GCAGTACTTCGCTCTCTTTCG GT27 AAACCGTCCTGGAAAACCA 84 kvgA GT28 CAACCAGCTGGATAGCATGA GT13 GTATTTTTATTCGCGATGTACTGC 67 kvhR GT14 GCCTGAACAGCGGAGAGA

Table 3. qRT-PCR analyses of the expression of iron acquisition genes in K.

1

pneumoniae wild-type and fur strains

2 3

Systems Gene

RNA expression ratioa

Reference fur/wild type

Fe3+

Ferrichrome fhuA 1.73±0.19 (Ferguson et al., 1998) Aerobactin iucA 2.42±0.18 (Chen et al., 2004)

Enterobactin fepA 2.11±0.18 (Nassif & Sansonetti, 1986)

fepB 2.25±0.20 (Nassif & Sansonetti, 1986)

entC 3.09±0.15 (Nassif & Sansonetti, 1986) Ferric citrate fecA 1.61±0.16 (Braun & Mahren, 2005)

fecE 1.69±0.26 (Braun & Mahren, 2005) Salmochelin iroB 6.28±0.98 (Chen et al., 2004) Heme hmuR 3.08±0.65 (Thompson et al., 1999) Fe2+

Ferrous iron feoB 4.08±0.35 (Cartron et al., 2006)

sitA 1.97±0.23 (Sabri et al., 2006)

a. Mean expression ratio of fur mutant relative to wild-type parental strain CG43S3 4

5 6 7

Fig. 1

(A)

rmpA rmpA2 rcsA rcsB kvgA kvhR

Relative amo unt of m RNA 0 100 200 300 400 CG43S3 [pRK415] in LB fur [pRK415] in LB fur [pfur] in LB CG43S3 [pRK415] in LB+Dip fur [pRK415] in LB+Dip

fur [pfur] in LB+Dip

3 4

(B)

(C)

Fig. 2 1 2 3 4 5

Glucuro

nic

acid

am

ount

(



g/10

cf

u)

0

10

20

30

40

50

CG43S3 rmpA rmpA2 rcsA CG43S3 rmpA rmpA2 rcsA

Fig. 3 1 (A) 2 3 4

(B) 1

2

3 4

(C) 1 2 3 4 5 6 7 8 9 10 11

Fig. 4 1 2 3 4 5 LB LB+DPP M9 M9+FeSO4

Glucuronic acid amount

(  g/10 9 cfu) 0 20 40 60 80 100 120 140 CG43S3 [pRK415] fur [pRK415] fur [pfur]

Fig. 5 1 (A) 2 3 4 (B) 5 6 7



fur

wild-type



fur

(pfur)



fur

(pRK415)

(C) 1

2 3

pT7-7

P

P

P

P

P