Fur蛋白質在克雷白氏肺炎桿菌CG43中的功能探討

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(1) . 國立交通大學. 生物科技研究所碩士論文. Fur 蛋白質在克雷白氏肺炎桿菌 CG43 中的功能探討 Functional characterization of Fur in Klebsiella pneumoniae CG43S3. 研究生：吳嘉怡學號 : 9528501 指導教授：彭慧玲博士 . 中華民國九十七年七月. .

(2) . 中文摘要在細菌生命週期中，具有足量的鐵離子是維持新陳代謝和生長必需的，因此多數細菌演化出同時擁有數個螯鐵系統。然而，過多鐵離子卻會造成氧化自由基形成，這對細菌本身將會造成致命的傷害，是故微生物體內的螯鐵系統都需要受到緊密的調控。在革蘭氏陰性菌中，許多參與螯鐵系統基因轉錄過程都受到 Fur 蛋白質的抑制。此外,除了調控鐵離子濃度在細菌體內外的平衡，當細菌遭受到酸性或過氧化環境壓力 Fur 蛋白質也參與啟動保護機制。克雷白氏肺炎桿菌 CG43 是一具有毒性的臨床分離株，被 K2 血清型莢膜所包覆。為了瞭解 Fur 在克雷白氏肺炎桿菌 CG43 中扮演的角色，我們建構了 fur 基因缺損株，此基因缺損明顯影響細菌生長。有趣的是，fur 突變株菌落相較於野生株顯得比較黏稠，透過莢膜多醣定量顯示 fur 突變株莢膜明顯增量；進一步分析啟動子活性，我們發現莢膜多醣合成基因組 orf3-15 表現量因 fur 基因缺損而增加；而 fur 突變株對小白老鼠的半致死劑量較野生株減少，顯示 Fur 蛋白質可能參與調控細菌的毒性。另外，我們也發現 fur 缺損使克雷白氏肺炎桿菌 CG43 對於酸性(pH 3)和過氧化 (H2O2 treatment)的環境壓力變得較為敏感。最後，fur 的缺損還會提高 iro、iuc 基因組(攝取三價鐵相關)以及 feo 基因組(攝取二價鐵相關)的啟動子活性，顯示 Fur 蛋白質在調控克雷白氏肺炎桿菌體內鐵離子平衡上扮演重要的角色。. i .

(3) . Abstract Bacteria have evolved several acquisition systems for sufficient quantities of iron to support their metabolism and growth. Iron overloading would lead to the formation of hydroxyl radicals and hence microorganisms have equipped a tight regulatory system for iron uptake. In Gram-negative bacteria, Fur protein represses the transcription of many genes that are involved in iron acquisition. In addition to control iron homeostasis, Fur also participates in protective responses to acid stress and oxidative stress. Herein, we report the construction of fur deletion mutant and found that the deletion impaired the growth of Klebsiella pneumoniae CG43S3, a highly virulent strain heavily encapsulated with K2 serotype. Interestingly, the deletion rendered the bacteria more mucoid phenotype which probably resulted from increasing amount of the glucuronic acid content. In addition, an increased activity of Pcps-orf3-15 was found. The deletion of fur slight reduced the LD50 using mouse lethality assay suggesting an involvement of Fur in virulence regulation. Moreover, fur deletion was found to increase the bacterial sensitivity to either acidic or oxidative stress. Finally, promoter activity measurement revealed that the fur deletion enhanced the activity of iro and iuc, respectively encoding enterobactin and aerobactin siderophore uptake systems, and feoABC, coding for ferrous iron uptake system. This indicated that Fur plays a major role for the regulation of iron acquisition system in the bacteria. ii .

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(5) . . Table of Contents . Abstract (Chinese) ..................................................................................... i Abstract .................................................................................................... ii Acknowledgment .................................................................................... iii Table content ............................................................................................. v Figure content ..........................................................................................vi Abbreviation ......................................................................................... viii Introduction ............................................................................................... 1 Materials and methods .............................................................................. 7 Results ..................................................................................................... 12 Discussion ............................................................................................... 18 References ............................................................................................... 22 Tables ...................................................................................................... 35 Figures ..................................................................................................... 39 . iv . .

(6) . . Tables Table 1.. Bacterial strains used and constructed in this study ................ 35. Table 2.. Plasmids used and constructed in this study ......................... 36. Table 3.. Primers used in this study ...................................................... 37. Table 4.. LD50 using mouse lethality assay .......................................... 38. . v .

(7) . Figures Fig. 1.. Comparison of fur organization and Fur protein sequence alignment ........ 39. Fig. 2.. Schematic representation of the fur deletion and the deletion mutant verified by PCR analysis ............................................................................................. 40. Fig. 3.. Fur gene deletion effect on the growth of K. pneumoniae CG43S3.............. 41. Fig. 4.. Fur gene deletion rendered a more viscous phenotype.................................. 42. Fig. 5.. CPS analysis of K. pneumoniae CG43S3 and K. pneumoniae CG43S3△fur. ......................................................................................................................... 43. Fig. 6.. CPS analysis of K. pneumoniaeCG43S3[pRK415] and K. pneumoniae CG43S3△fur[pfcpRK]. .................................................................................. 44. Fig. 7.. Deletion effect of fur on the expression of CPS biosynthesis. ...................... 45. Fig. 8.. Deletion effect of Fur on the expression of rmpA, rmpA2, rcsB and kvgA ... 46. Fig. 9.. Deletion effect of fur on the acid stress response ........................................ 47. Fig. 10.. Deletion effect of fur on the sensitivity to H2O2 ......................................... 48. Fig. 11.. Deletion effect of fur on the expression of iro system .............................. 49. Fig. 12.. Deletion effect of fur on the expression of iuc system. ............................... 50. Fig. 13.. Deletion effect of fur on the expression of feo system .............................. 51. Fig. 14.. Deletion effect of Fur on the autoregulation of fur. .................................... 52 vi . .

(8) . Fig. 15.. Schematic representation of the feoB deletion and verified by PCR analysis ....................................................................................................................... 53. Fig. 16.. FeoB gene deletion effect on the growth of K. pneumoniae CG43S3. ....... 54. Fig. 17.. FeoB gene deletion effect on the sensitivity to streptonigrin. ..................... 55. vii .

(9) . Abbreviation. BLAST. basiclocal alignment search tool. bp. base pair. CFU. colony forming unit. CPS. capsular polysaccharide. DNA. deoxyribonucleic acid. IPTG. isopropyl-lthio-β-D-galactopyranoside. LB. Luria-Bertani. ORF. open reading frame. ONPG. ο-nitrophenyl-β-D-galactopyranoside. PCR. polymerase chain reaction. rpm. revolutions per minute. . . viii .

(10) . . Introduction Iron is essential for growth Iron is essential for the growth of nearly all organisms. It could be a cofactor of many enzymes including ribonucleotide reductase, RNA polymerase Ⅲ, various amino acid hydroylases and dioxygenases, and the enzymes, such as superoxide dismutase, catalase and peroxidase, participate in oxygen metabolism in microbial and mammalian cells (83). Iron is also necessary for the activities of cytochromes, hydrogenase, ferridoxin and succinate dehydrogenase that are involved in electron transfer. In vertebrates, T- and B-lymphocyte activity and natural killer cell function are all dependent on iron (11, 12). Iron exists as oxidized (Fe3+) and reduced form (Fe2+). Ferric iron has lower solubility and ferrous iron participates in Haber-Weiss-Fenton chemistry ( H2O2+Fe2+ →Fe3++OH-+OH·) that causes potentially cell damage (81). Hence, higher organisms have evolved mechanisms to lower the levels of free iron. In human body, the majority of iron is intracellular, either complexed with metalloproteins such as haemoglobin (74.3%), myoglobin (3.3%), catalase (0.11%) and cytochrome C (0.08%), or stored in the iron-storage protein ferritin (16.4%) and its insoluble degradation product haemosiderin (21, 74). In blood, tissues, and tissue fluids, there are concentrations of iron sufficient to supply the needs for cellular metabolism. The 1 .

(11) . iron-binding protein including transferrin and lactoferrin take charge of the iron transport and recycling. Thus, usable iron in human host is below that required for microbial growth. Bacteria have evolved several systems for the acquisition of sufficient quantities of iron to support their metabolism and growth. These include production of powerful iron-binding compounds (siderophores), direct utilization or uptake host iron-binding proteins, reduction of the Fe3+ insoluble form to soluble useful form of ferrous iron (Fe2+), enzymatic degradation of iron-binding compounds, production of lethal compound (exotoxins) that may eliminate competitors for usable iron resources (77). For instance, E. coli strains harboring the plasmid ColV-K30 were reported to own two independent siderophore systems, enterochelin and aerobactin (82, 80). Neisseria meningitidis (24), Haemophilus influenzae (65), Vibrio cholerae (69) and Campylobacter jejuni (64) use host iron compounds, heam and heamoglobin. Listeria monocytogenes (16), Pseudomonas aeruginosa (17), Bifidobacterium bifidus (7) and Streptococcus mutans (28) were reported to reduce ferric iron at cytoplasmic membrane and subsequently transport ferrous iron into the cytoplasm. To avoid iron overloading that would lead to the formation of hydroxyl radicals, microorganisms have tight regulation of iron uptake. In Gram-negative bacteria, iron regulation is mediated by the Fur protein, which represses the transcription of many 2 .

(12) . genes that are involved in iron acquisition across a wide range of species (29). Some bacteria have also evolved mechanism whereby gene transcription is initiated by the availability of iron-loaded siderophores on the cell surface. The best known of these systems is the ferric citrate system in E. coli (25). In addition, a global response to iron limitation has been observed at the level tRNA modification (36) observed in Salmonella typhimurium, K. pneumoniae, P. aeruginosa and N. meningitidis (56, 2).. Klebsiella pneumoniae K. pneumoniae is a gram-negative, nonmotile, encapsulated rod-shaped bacterium of the family Enterobacteriaceae. It is an opportunistic pathogen, attacking immunocompromised and hospitalized patients suffering severe diseases. K. pneumoniae causes community acquired and nosocomial infections, including septicemia, pneumonia, urinary tract infection, meningitis, and purulent abscess at various sites (84). Klebsiella usually have well developed polysaccharide capsules, which make their colonies characteristic mucoid. Capsules that form thick bundles of fibrillous structures covering the bacterial surface in massive layers is the essential virulence factor. This could protect the bacterium from phagocytosis by polymorphonuclear granulocytes, and prevents killing by bactericidal serum factors (12). Acording to the diverse structures of capsular polysaccharide, K. pneumoniae could be classified into 3 .

(13) . 77 serological K antigen types (45). K1 and K2 strains were found to be especially virulent assessed with the mouse peritonitis model (58). Beside the capsular polysaccharide, lipopolysaccharide, adhesin, iorn-acquisition system, and serum resistance factors are also major virulence factors involved in K. pneumoniae infections (66). Multiple siderophore biosynthesis systems have been reported in K. pneumoniae, which include enterobactin, aerobactin and yersiniabactin; respectively encoded by iro, iuc and ybt gene clusters (47). We have previously identified two siderophore biosynthesis gene clusters, iro and iuc, on the large virulence plasmid pLVPK in K. pneumoniae CG43, a highly virulent clinical isolate of K2 serotype (14). Besides the ferric iron uptake systems, two ferrous iron uptake systems, feoABC and sitABCD, could. be. identified. in. the. genome. of. K.. pneumoniae. MGH78578. (http://www.ncbi.nlm.nih.gov/sites/entrez).. Fur In 1978, Ernst et al., (26) described a mutation in S..enterica Typhimurium, designated fur (ferric uptake regulator), that resulted in constitutive expression of several high-affinity iron assimilation systems. Fur, a 17-kDa polypeptide, negatively regulates the iron acquisition systems in most gram negative bacteria. (3, 51) In iron replete condition, Fe+2-Fur binds to the promoter of iron-regulated genes thereby 4 .

(14) . prevents their expression. Under low iron, Fur is present in the iron-free form, which does not bind to the regulated promoters (3). Fur not only controls iron homeostasis by regulating the promoters of iron uptake systems directly, but also represses a small RNA, RyhB, that in turn negatively regulates the expression of iron-rich enzymes such as succinate dehydrogenase, fumarase, and aconitase. This allows the production of these enzymes to be activated in response to available iron (55). The N-terminal domain of Fur is involved in DNA binding and the C-terminal domain in dimerization (72). A consensus sequence has been derived for the Fur binding site in E coli, and this is referred to as an iron or Fur box (34). The palindromic sequence GATAATGATAATCATTATC has been elucidated for different bacteria (62, 85). In addition to the role is in the regulation of iron uptake systems, its involvement in regulation of acid tolerance response, oxidative stress response, some metabolic pathways, and expression of virulence factors has been proposed (27). In some of these cases, Fur acted positively rather than negatively in the regulation of the expression of certain genes (61, 37, 23). For example, fur deletion resulted in decrease of oxidative stress response and increase of DNA damage in E. coli (75). In S. enterica Typhimurium, fur mutation rendered an acid-sensitive phenotype (30). In H. pylori, Fur is also involved in acid resistance (8). In response to iron, Fur is often 5 .

(15) . but not always involved in autoregulation in many Gram-negative bacteria (19, 1, 53).. FeoABC Feo system was described for the first time in E. coli in 1987 (38). The ferrous iron acquisition system has been experimentally identified in seven additional microbes including Porphyromonas gingivalis (18), Leptospira biflexa (52), Helicobacter pylori (78), Shigella flexneri (68), Salmonella enterica serovar Typhimurium (10), Legionella pneumophila (67), and the cyanobacterium Synechocystis sp (44.). In Campylobacter jejuni, FeoB-mediated ferrous iron acquisition has been found to play an essential role in bacterial virulence (60). Feo consists of FeoA, a 75-residue hydrophilic protein probably required to sense the concentrations of iron; FeoB, an integral cytoplasmic membrane protein of 773 amino acids required for ferrous iron acquisition; FeoC (YhgG), a small protein (75 amino acid) with unknown function (43, 54). In E. coli, Fur and Fnr binding sites were found in feo promoter, and both global regulators have been shown to regulate the expression of Feo system (13).. . . 6 .

(16) . Materials and methods. . Bacterial strains, and growth conditions The bacterial strains and plasmids used in the present study are described in Table 1. K. pneumoniae CG43 is clinical isolated strains from Chang Gung Memorial Hospital Linkou branch. E. coli and K. pneumoniae were cultured aerobically at 37 ℃ in Luria-Bertani (LB) broth or on LB agar plates supplied with appropriate antibiotics. The concentrations of antibiotics added in mediums include streptomysin (500 μg/ml), ampicillin (100 μg/ml), chloramphenicol (35 μg/ml), kanamycin (25 μg/ml), and tetracycline (5 μg/ml).. . Construction of gene-deletion mutants and complement strains The fur and feoB deletion strains were made by allelic exchange. Approximately 1000 bp sequences flanking both sides of the deleted region were cloned into plasmid pKAS46 (49), a suicide vector containing rpsL, which allows positive selection with streptomycin for loss of the vector, to generate an in frame deletion plasmid. The resulting plasmids were then mobilized to K. pnumoniae CG43S3 through conjugation from E. coli S17-1 λpir. The transconjugants, carrying with constructed plasmid integrated in the chromosome via homologous. 7 .

(17) . recombination, were selected by ampicillin and kanamycin on minimal medium.one of colonies was grown in LB at 37 ℃ for overnight and then spread onto a LB plate containing 500 μg/ml streptomysin. The streptomysin-resistant and kanamycin sensitive colonies were selected and the deletion of gene verified by PCR. The primer pairs used for PCR amplification are listed in Table 3. To construct the complement strains (Table2), the target gene including the promoter and coding sequences was amplified with the specific primers, the PCR product cloned to yT&A, and then subcloned to pRK415.. . Quantification of CPS CPS was extracted by using the method described (22). Briefly, bacteria were collected from 500 μl of culture medium and mixed with 100 μl of 1% Zwittergent 3-14 detergent (Sigma-Aldrich, Milwaukee, WI) in 100 mM citric acid (pH 2.0). The mixture was incubated at 50℃ for 20 min. After centrifugation, 250 μl of the supernatant was transferred to a new tube and CPS was precipitated with 1 ml of absolute ethanol. The pellet was then dissolved in 200 μl distilled water and a 1,200 μl of 12.5 mM borax (Sigma-Aldrich, Milwaukee, WI) in H2SO4 was added. The mixture was vigorously vortexed, boiled for 5 min, cooled, and then 20 μl 0.15% 3-hydroxydiphenol 8 . .

(18) . (Sigma-Aldrich, Milwaukee, WI) was added and the absorbance at 520 nm was measured. The uronic acid content was determined from a standard curve of glucuronic acid (Sigma-Aldrich, Milwaukee, WI) and expressed as μg per 109 CFU.. . Survivals under acid stress According to the described method with some modification (37),. the. bacteria were grown overnight in LB and 1/20 of the bacteria refreshed grown for 3 h. The cultures were then transferred to pH 5.8 or pH 4.4 LB broth (adjusted with HCl) for acid adaptation 1 h. Finally, the cultures were subjected to pH 3 acid stress for 1 h and then plating onto LB agar for viable counts. Data shown are from the representative experiment performed triplicately.. . Survival rate of oxidative stress As described with some modification (75), the bacteria grown in LB medium at 37℃ overnight were diluted in 1/20 and refreshed grown for 3 h, and H2O2 from 0 to 30 mM was added. After 20 min of incubation by shaking at 37℃, the viable bacteria were determined by plating the cultures onto LB plates.. . Streptonigrin resistance 9 . .

(19) . As the described method (67), the bacteria were grown in LB broth for 16 h and 100 μl of the bacteria in 10-fold serial dilution were plated on LB agar containing either 0.75, 1.5 or 3 μM streptonigrin. . β-galactosidase activity assay β-galactosidase was assayed according to the method of Miller (57). The bacteria in the early or late logarithmic growth phase (optical density at 600 nm 0.4 or 0.7) were taken 100 μl, and mixed with 900 μl Z buffer (60 mM Na2HPO4, 40 mM NaH2PO4, 10 mM KCl, 1 mM MgSO4, 50 mM β-mercaptoethanol), 17 μl of 0.1% SDS and 35 μl chloroform and incubated for 10 min at 30℃. Subsequently, 200 μl of 4 mg/ml ο-nitrophenyl-β-D-galactopyranoside (ONPG) was added and the mixture vortexed for 10 s, then incubated at 30℃ until yellow color was apparent. Finally, the reaction was stopped by adding 500 μl of stop solution (1 M Na2CO3) and the absorbance of the supernatant was measured OD420. One unit of β-galactosidase is defined as the hydrolysis of 1 nmol ONPG per min per mg protein.. . Mouse lethality assay The 4~5-week-old female Balb/c mice were obtained from National 10 . .

(20) . Laboratory Animal Center and were acclimatized in an animal house for 1 week. The tested bacteria were cultured at 37℃ for 16 h in LB broth. Four mice of a group were injected intraperitoneally with bacteria resuspended in 0.2 ml PBS. The 50% lethal doses, based on the number of survivors after 10 days, were calculated by the method of Reed and Muench (59) and expressed as colony forming units (CFU).. 11 .

(21) . Results. . Sequence analysis of K. pneumoniae Fur. In comparison of the Fur amino acid sequences of K. pneumoniae CG43, Escherichia coli K12, and Shigella flexneri 5b (http://www.ncbi.nlm.nih.gov /GenBank) as shown in Fig. 1B revealed 94% identityl feature. Analysis of the genomic location also revealed a conserved organization with fur gene in the middle, upstream with fldA, coding for a putative citrate-proton symporter, and Flavodoxin 1 encoding gene citA at the downstream (Fig. 1A).. . Construction of fur deletion muatnt. As shown in Fig. 2A, 454 bp of the fur coding sequence was designed to be deleted by the allelic exchange method. The PCR products of 1743 bp and 1289 bp obtained in Fig. 2B using the same primer pair on different templates DNA from K. pneumoniae CG43S3 and the selected fur mutant demonstrated the deletion. The fur deletion appeared to negatively affect the growth in LB or M9 broth (Fig. 3A & 3B). Introducing the plasmid pfcpRK, pRK415 carrying fur gene, into the mutant was found to compensate the deficiency.. 12 .

(22) . . Fur is involved in the regulation of CPS biosynthesis. Interestingly, the mutant strain displayed larger and more glistening colony morphology than the wild type on LB agar (Fig. 4A). Introduction of pfcpRK into the fur mutant restored the phenotype (Fig. 4B). When the bacteria cultures were subjected to low-speed centrifugation, the mutant were precipitated much slower than its parental strain K. pneumoniae CG43S3 (Fig. 5A). Subsequently, the CPS production in CG43S3 and the mutant were quantified by measuring the glucuronic acid content, the core component of the K2 CPS. As shown in Fig. 5B, the deletion of fur enhanced the CPS synthesis. The transformation of the fur mutant with the plasmid pfcpRK decreased the sedimentation rate (Fig. 6A) and CPS production (Fig. 6B). As shown in Fig. 7A, biosynthesis of K. pneumoniae CG43 K2 CPS has been shown to be determined by three major transcripts namely orf 1-2, orf 3-15, and orf 16-17 (88). The promoter activity measurement as shown in Fig. 7B, deletion of fur had positive effect on the expression of cps clusters orf 3-15 and orf 16-17. However, no apparent effects on the promoter activities of RmpA, RmpA2, RcsB, and KvgA, the regulators of mucoid phenotype, were noted (Fig. 8).. 13 .

(23) . . Effect of fur deletion on the acid sensitivity If K. pneumoniae Fur is involved in acid stress response as the Fur reported for many other bacteria (37, 39, 70, 73) is also investigated. As shown in Fig. 9, fur deletion had negative effect on the bacterial survivals at pH 3 regardless the adaptation at pH 5.8 or pH4.4. The introduction of the fur-expression plasmid pfcpRK appeared to restore the viability.. . The fur deletion reduced the bacterial survival upon H2O2 treatment Hydrogen peroxide causing DNA damage via the Fenton reaction had been demonstrated. E. coli Fur has been associated with the bacterial survivals with H2O2 treatment (75). To know if Fur provides protection in K. pneumoniae under H2O2 treatment, different concentrations of H2O2 were added to the cultures of the wild type bacteria, △fur muatnt, △fur [pRK415], and △fur [pfcpRK]. While the concentration of H2O2 reached to 20 mM, fur deletion had apparent effect on the bacterial survivals. As shown in Fig. 10, an increased sensitivity to H2O2 of the mutant was observed and the complementation with pfcpRK was able to restored the survivals.. 14 .

(24) . . A decreased LD50 of the fur mutant In many pathogenic bacteria, deletion of fur causes virulence reduction (46). To know whether Fur affects the virulence of K. pneumoniae CG43S3, LD50 of the mutant using mouse lethality assay was determined. As shown in Table. 4, LD50 of the Fur deletion mutant slightly decreased.. . Fur regulates the expression of the iron acquisition systems, iro, iuc and feo. In many bacteria, iron acquisition was regulated in part by the Fur protein (15). The effect of fur deletion on the expression of the iron acquisition systems including iro, iuc, and feoABC, all carrying a typical Fur box on the putative promoters (respectively Fig. 11A, 12A, and 13A), were examined. As shown respectively in Fig. 11B, 12B, and 13B, the putative promoter activity of iro, iuc, and feo were determined using LacZ as promoter reporter. As shown in Fig. 11C, 12C, and 13C, the activity of Piro, Piuc or Pfeo apparently enhanced in the fur deletion mutant. Although a consensus Fur box could be identified upstream of the fur gene (Fig. 14A), the deletion of fur had no effect on the expression of fur using the promoter activity measurement (Fig. 14 B and C).. . Sequence analysis of feoABC and construction of feoB deletion mutant 15 . .

(25) . In general, Feo system consists of at least two genes, feoA and feoB, which encode, respectively, an iron-sensing protein and an iron permease. In some organisms, an additional locus, feoC, is present immediately downstream (13). Amino acid sequence comparison with E. coli K12 FeoA, FeoB, and FeoC revealed identity of respectively 81%, 67%, and 69%. To investigate if Feo system plays a major role in ferrous iron uptake in K. pneumoniae, feoB deletion mutant was constructed. The entire FeoB coding sequence of 2316 bp as shown in Fig. 15A was deleted and the deletion in the selected mutant verified by PCR (Fig 15B). To investigate the feoB deletion effect, growth of K. pneumoniae CG43S3 and the feoB deletion mutant were monitored in LB broth and LB broth loaded with ferrous iron. As shown in Fig. 16A and B, no obvious difference could be observed between the wild type and the feoB mutant in LB or the medium loaded with 50 μM ferrous iron. The deletion effect on the resistance to streptonigrin was also investigated. This antibiotic, streptonigrin, possesses an iron-dependent toxicity and has been used to select E. coli FeoB mutant (67). As shown in Fig. 17, addition of 0.75 μM to 3 μM of streptonigrin had no apparent difference for the survivals between K. pneumoniae CG43S3 and the feoB mutant. . LD50 of the feoB mutant using mouse lethality assay 16 . .

(26) . The LD50 determined as shown in Table 4 indicated that the deletion of feoB does not cause apparent effect on the virulence of K. pneumoniae.. 17 .

(27) . Discussion. Fur protein was named as “ferric iron uptake regulator”, which directly points out the tight correlation of this protein with iron uptake. However, more and more recent articles indicate that Fur plays roles not only in iron uptake, but also others functions including acid tolerance in S. typhimurium and S. flexneri (76, 9), and oxidative stress response in E. coli and H. pylori (75, 79). Similar to the report in Pseudomonas aeruginosa (77), the deletion of fur appeared to retard the growth. While the changes of colony phenotype to more sparkling and transparent has not been mentioned. In addition to the reported functions, our results add to a novel role of Fur which is a negative regulator for the expression of the cps genes. Analysis the promoter sequences of the cps-orf 3-15 and cps-orf 16-17, it revealed a consensus Fur box on the Porf 3-15, but not on Porf16-17. Although Fur box was identified for promoters of RmpA, RmpA2, and RcsB, there was no distinct difference in the LacZ activity assay between the wild type and the Fur gene mutant. Besides, there was no Fur box on PkvgA, and no effect of Fur deletion on PkvgA activity was observed. pH homeostasis is the process whereby a cell maintains a relatively constant intracellular pH over a broad range of external values. The acid tolerance response 18 .

(28) . (ATR), one of mechanisms that keep pH homeostasis, has been discovered in S. typhimurium and E. coli (77). The acid tolerance response is triggered in Salmonella species at pH values between 6.0 and 5.5, but protects bacteria against much stronger acid (pH 3 to 4.0) (32). In S. typhimurium, the fur gene product has been implicated to contribute a key regulatory function to the acid tolerance response (32). Furthermore, Fur has been shown to participate in responses to low pH in S. flexneri (41) and H. pylori (35). In K. pneumoniae CG43S3, the deletion of fur reduced the survivals under acid treatment suggesting a positive regulatory role in the acid stress response. The permanent derepression of iron assimilation system in Fur mutant could produce oxidative stress leading to various cell damages. E. coli fur mutant has been shown to be sensitive to hydrogen peroxide, increased oxidative DNA damage, and mutations under aerobic conditions (75, 86). In H. pylori, Fur was also proved as a key role in antioxidant systems (79). As shown in Fig. 10, following the increasing doses of hydrogen peroxide, from 10 mM to 30 mM, the survival rate of the fur mutant decreased more sharply than the wild type and the mutation effect could be complemented by introducing a fur-expression plasmid. This indicates that Fur protein probably also acts a positive role in the oxidative stress response. Deletion of fur caused attenuated virulence in S. flexneri (63) and A. tumefaciens (46), and a competitive defect in colonization in H. pylori (35). To assess 19 .

(29) . the role of Fur in K. pneumoniae, LD50 using mouse lethality assay of the mutant was measured. Although the fur deletion mutant appeared to grow more slowly than the wild type, a slight reduction of LD50 was observed. The iroBCDN gene cluster found on the virulence-associated plasmid pLVPK is responsible for enterochelin synthesis. In S. enteric, a typical Fur DNA binding site on the iro promoter region is required for the regulation of Fur on the expression of iro gene cluster (4, 5). The iucABCD involved in the biosynthesis of aerobactin is transcriptionally regulated by Fur in E. coli (20). In K. pneumoniae NTUH2044, iucABCDiutA was shown to be significantly prevalent in PLA-associated (pyogenic liver abcess) isolates (KP-PLA) (40). The iucA in K. pneumoniae CG43S3 has been reported as an IVE gene (48). In either Piro, Piuc, or Pfeo, putative Fur box could be found. The LacZ activity measurement further supports that the regulation of iro, iuc, or feo by Fur is probably via direct binding to the Fur box. These indicated that Fur is a major regulator for iron acquisition in K. pneumoniae. Autoregulation of Fur has been described in K. pneumoniae (1), and a putative Fur box was predicted on the fur promoter. However, the LacZ activity measurement revealed no apparent regulation. The presence of multiple iron transport system in K. pneumoniae suggests that iron acquisition systems are needed during infection. The PLA-associated strains were shown to be able to use wider range of iron sources than non-PLA-associated strains 20 .

(30) . (40). Whereas, obvious negative effect of feoB deletion suggested another ferrous iron uptake system SitABCD plays more important role in K. pneumoniae. The possibility could be demonstrated by generation of sitBCD deletion mutant. It is also possible that the activity of other ferric iron uptake system acts to compensate the deficiency of feoB.. 21 .

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(44) . . Table 1.. Bacterial strains used and constructed in this study . Strain. Genotype or relevant property. Reference or source. E. coli: RecA1 supE44 endA1 hsdR17 gyrA96 rolA1 thi △. JM109. (lac-proAB). S17-1λpir. Laboratory stock. Tpr Smr recA, thi, pro, hsdR－M＋ [RP4-2-Tc::Mu:KmrTn7] (pir). De Lorenzo et al., 1994. K. pneumoniae: CG43 CG43-S3. Clinical isolate of K2 serotype △rspl, Smr. Laboratory stock. Z01. CG43-S3△lacZ Smr. Laboratory stock. △fur. CG43-S3△fur Smr. This study. △feoB. CG43-S3△feoB Smr. This study. Z01△fur. r. Z01△fur Sm. This study. 35 . Laboratory stock.

(45) Table 2.. Plasmids used and constructed in this study. Plasmids. Relevant characteristic. Source or reference. yT&A vector pKAS46 pRK415 pLacZ15. PCR cloning vector, Apr Suicide vector, Apr Kmr Shuttle vector, mob+, Tcr A derivative of pYC016 (87), containing lacZ as a reporter, Cmr. pfur2K46 pfeoB2K46 pfcpRK. pKAS46 carrying a △fur fragment pKAS46 carrying a △feoB fragment A 843 bp PCR product of the fur locus with the putative promoter cloned into pRK415. Sigma Novagene Laboratory stock Laboratory stock This study This study This study. pirocyyT. A 455 bp PCR product of the iro putative promoter region cloned into yT&A. This study. piuccyyT. A 721 bp PCR product of the iuc putative promoter region cloned into yT&A. This study. pfeoyT. A 564 bp PCR product of the feo putative promoter region cloned into yT&A. This study. pfuryT. A 426 bp PCR product of the fur putative promoter region cloned into yT&A. This study. pirocyZ15. A BamHI/BglII fragment of pirocyyT cloned into the pLacZ15. This study. piuccyZ15. A BamHI/BglII fragment of piuccyyT cloned into the pLacZ15. This study. pfeoZ15 pfurZ15. A BamHI/BglII fragment of pfeoyT cloned into the pLacZ15 A BamHI/BglII fragment of pfuryT cloned into the pLacZ15. This study This study. pRmpAZ15. A 0.5 kb fragment of the rmpA putative promoter region cloned into the pLacZ15. Laboratory stock. pRmpA2Z15. A 0.5 kb fragment of the rmpA2 putative promoter region. Laboratory stock. cloned into the pLacZ15 pRcsBZ15. A 0.4 kb fragment of the rcsB putative promoter region cloned into the pLacZ15. Laboratory stock. pKvgAZ15. A 0.2 kb fragment of the kvgA putative promoter region cloned into the pLacZ15 A 0.8 kb fragment of the cps orf1-2 promoter region cloned into the pLacZ15. Laboratory stock. porf3-15Z15. A 0.9 kb fragment of the cps orf3-15 promoter region cloned into the pLacZ15. Laboratory stock. porf16-17Z15. A 0.4 kb fragment of the cps orf16-17 promoter region cloned into the pLacZ15. Laboratory stock. porf1-2Z15. 36 . Laboratory stock.

(46) . . Table 3. Primers used in this study. Primer. Sequence. CY001 CY002 CY003 CY004 CY007 CY008 CY009 Pfeo01 Pfeo04 Pfeo05 Pfeo06 Pfeop01 Pfeop02 Piucp01 Piucp02 Pirop01 Pirop02. 5'-GAATTCTGCTGATGACCCAGTTAACC-3' 5'-GGATCCGTTGTCAGTCATGCGGAATC-3' 5'-GGATCCACGCGGTGGAAACATAATTC-3' 5'-GAATTCACCTCTGGGAGAACGACAATG-3' 5'-TCTAGAGGCAGGTTGGCTCTTCAGTC-3' 5'-GGATCCATGAAGACAGCCAGCCGGA-3' 5'-AGATCTGCCCAGCCTTCTTTAATGCGG-3' 5'-GAATTCTCACCAACGTCACCAACTTC-3' 5'-TCTAGACAAACCATGGGCACAGAGA-3' 5'-GGTACCGTGGTCTTGCTGGAGTTAGG-3' 5'-GGTACCTGCCACTAAGGAGGGACTGT-3' 5'-GGATCCCAACAGCGCGATGATGGAT-3' 5'-AGATCTGCCAGCATGCCGAGGGAGA-3' 5'-GGATCCAGAGGGTGATTTGCCAGCAT-3' 5'-AGATCTGGAAGCACTGAGCAGCCACA-3' 5'-GGATCCGATTTCAGTACGGCATGGAC-3' 5'-AGATCTACGGGAAACGCCTGTGCCA-3'. . . 37 .

(47) . . Table 4. LD50 using mouse lethality assay. strain. CG43S3. LD (cfu). 1.33×10. 50. . Δfur. 4. 3. 8.8×10. . 38 . ΔfeoB 4. 1.35×10.

(48) . (A) . (B) . . F Fig. 1. (A A) The com mparison of o genes organization o n along with w fur (B B) Fur Protein sequ uence alignment. Sequencess shown arre Klebsellaa pneumoniiae CG43 Fur, Escheerichia coli K-12 Fur,, and S Shigella fleexneri Fur (http://www ( w.ncbi.nlm.nnih.gov /GeenBank). Thhe putative regulatory Fe-sensingg site r residues (S1) are indicated with ciircles, and the t structuraal Zn-bindinng site residdues (S2) arre indicated with t triangles.. 39 .

(49) . . . Figg. 2. Schem matic reprresentation of the fu ur deletion (A) and the deletio on verifiied by PCR R (B). M: marker. m Lanee 1: K. pneumoniaeCG443S3. Lane 2: K. pneumoniaeC CG43S3△fuur.. 40 .

(50) . (A) . (B B) . Grrowth curvve in LB. Groowth curvve in M9. Fig. 3. 3 Fur genee deletion effect on the t growth h. K. pneum moniae CG G43S3 (bluee line), fur deletion mutant m (triaangle line) and a fur com mplement (square linee) were grow wth in LB broth (A) or M9 broth (B). The concentratio ons of antibbiotics addeed in medium ms include streptomyysin (500 μg/ml) μ for K. pneum moniae CG443S3 and ffur deletio on mutant; tetracyclinne (5 μg/ml)) for fur com mplement.. 41 .

(51) . A) (A. . Δfur. W Wild type. (B) . Δfu ur-pRK415. Δ Δfur-pfcpRK K. F Fig. 4. Furr gene deletion rend dered a mo ore viscouss phenotyp pe. The baccteria weree s streaked onn SOC agarr (42) at 377℃, 20 h. The conceentrations oof antibioticcs added inn m mediums innclude streptomysin (5000 μg/ml) for f K. pneumoniae CG G43S3 and fur f deletionn m mutant; tetraacycline (5 μg/ml) for Δfur-pfcpR Δ RK and Δfur-pRK415. 42 .

(52) . . . (B) . W type Wild. Δfu ur. Fig. 5. CPS analyysis of K. pneumonia p ae CG43S3 3 and K. pneumoniae p e CG43S3△ △fur usingg sedimentaation assay (A), and glucuronic g acid measu urement (B B). The gluccuronic acidd contents were w determ mined afteer the baccteria were incubatedd in LB broth b addedd streptomyssin (500 μg//ml) for 16 h. h. 43 .

(53) . . (A) . . (B) . Fig. 6. 6 CPS analysis a o K. pn of neumoniae CG43S3--pRK415 and K. pneumoniaae CG43S3△ △fur‐pfcpR RK using sedimenttation assay (A), and glucu uronic aciid measurem ment (B). The T glucuronic acid con ntents weree determinedd after the bacteria b were incubatedd in LB brothh added tetrracycline (5 5 μg/ml) forr 16 h.. 44 .

(54) . . . (A) . (B B) . Fig. 7.. Deletion effect e of Fu ur on the expression e of o CPS gen nes. Schemaatic represeentation of the CPS genes g prom moters of K. pneumonia ae CG43 (888) (A), and the activity y of Porf1-2, Porf3-15 annd Porf16-17 (B). ( The proomoter actiivities were determinedd for 1/20 dilution d of the overnnight grownn bacteria suubjected to refreshed r grrown for 6 hh.. 45 .

(55) . (A) . (B) . (C)) . (D) . Fig. 8. Deletion D eff ffect of Furr on the exp pression off rmpA, rmppA2, rcsB aand kvgA. The T activityy o PrmpA (A)), PrmpA2 (B)), PrcsB (C) and of a PkvgA (D D) were determined aftter the overn night grownn b bacteria in 1/20 1 dilutionn suspendedd in LB and d refreshed grown g for 6 h.. 46 .

(56) . . Fig. 9. Deletion efffect of furr on the accid stress response. r W type (bblack bar), Fur deletioon Wild mutant (tw will bar), furr complemeent strain (w white bar) annd pRK4155 containing g strain (graay bar) were grown g to mid-log m phasse, adapted at pH 5.8 (1) or pH 44.4 (2) for 1 h and theen transferred to pH 3 forr 1 h.. 47 .

(57) . . F 10. Deeletion effecct of fur on Fig. n the sensittivity to H2O2. Survivvals of wild type (black k bar), Fur muutant (twill bar), b fur com mplement sttrain (whitee bar) and pRK415 conntaining. sttrain ( gray. barr) were meaasured afterr 1/20 dilutiion of the overnight o grrown bacterria refreshed d grown in LB B for 3 h foollowed byy 20 min off challenge with 10 mM, m 20 mM M and 30 mM m H2O2, respectively.. 48 .

(58) . (A) . (B) . (C) . Fig. 11. Deletion n effect of Fur F on the expression n of iro systtem. (A) Thhe upstream non-coding g sequencess of iro and a the putative Fur boxx are shownn. (B) Schem matic repressentation off the Piro–laccZ fusion plasmidd. (C) LacZ activity meeasurement.. The activitty of Piro waas determinned after thee overnight grown bacteria b in 1/20 dilutioon suspended in LB and d refreshed grown for 8 h. 49 .

(59) . (A A) . (B B) . (C) . Fiig. 12. Deleetion effect of Fur on the t expresssion of iuc system. s (A)) The upstreeam non-cod ding sequennces of iuuc and the putative p Fur box are shoown. (B) Scchematic reppresentationn of the Piuc –lacZ fusioon plasm mid. (C) LaacZ activity measuremeent. The acttivity of Piucc was determ mined after the t overnigght grow wn bacteria in 1/20 diluution suspennded in LB and refreshed grown foor 8 h.. 50 .

(60) . . (A) . . (C) . (B B) . Fig. 13. Deletioon effect of o Fur on the expreession of feo fe system.. (A) The upstream non-codding sequennces of feo and the pu utative Fur box are shhown. (B) Schematic S represenntation of thhe Pfeo –lacZ Z fusion plaasmid. (C) LacZ L activitty measurem ment. The activity of Pfeo wass determineed after the overnight grown g bactteria in 1/20 0 dilution suspendded in LB annd refreshedd grown forr 8 h. 51 .

(61) . (A) . . (B) . (C C) . F 14. Deeletion effecct of Fur on Fig. n the autorregulation of o fur. (A) The upstreaam non-cod ding sequencces of fur and thhe putative Fur box arre shown. (B) ( Schemaatic represeentation of the Pfur –laacZ Z activity measuremen m nt. The activvity of Pfur was determ mined after the fussion plasmid. (C) LacZ overnight grow wn bacteriaa in 1/20 diluution suspended in LB and refreshhed grown for f 8 h.. 52 .

(62) . . . Fig. 15. Scchematic representatiion of thee feoB deletion (A) FeoB gene deeletion muttant was verified v by y PCR. (B B) M: markker. Lane 1: 1 CG G43S3. Lanne 2: CG43S S3△feoB.. 53 .

(63) . . . (A). (B B). Fig. 16. FeoB genee deletion effect on the growth of K. pneu umoniae C CG43S3. Wild W type (blue line), feoB mutannt (dotted liine) were growth g in LB B broth (A)) or LB add ded with 50 μM ferroous iron andd 50 μM asccorbate (B).. 54 .

(64) . . Fig. 17. FeoB genee deletion effect e on th he sensitivitty to strepttonigrin. Survivals off the wild tyype (black barr) and the FeoB F mutaant (white bar) b were measured aafter the baacteria grow wn overnight in i LB with the t indicateed concentraation of streeptonigrin.. 55 .

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