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To monitor trends in the distribution of yeast species and the susceptibilities of these species to commonly prescribed antifungal drugs, we conduct the Taiwan Surveillance of Antimicrobial Resistance of Yeasts (TSARY) every 4 years. We found that 25 of 294 Candida tropicalis isolates from TSARY 2014 and 31 of 314 C. tropicalis isolates from TSARY 2018 were resistant to fluconazole. We determined the genetic relatedness among fluconazole-resistant C. tropicalis isolates by multilocus sequence typing (MLST). Among 174 C. tropicalis isolates, including all 56 fluconazole-resistant, all 26 susceptible-dose dependent and 92 selected fluconazole-susceptible isolates, 59 diploid sequence types (DSTs) were identified. We found that 22 of the 25 fluconazole-resistant C. tropicalis from TSARY 2014 and 29 of the 31 fluconazole-resistant C. tropicalis from TSARY 2018 were genetically related and belonged to the same cluster (clade 4). A combination of mutation and overexpression of ERG11, encoding the target of azole drugs, was the major mechanism contributing to drug resistance. Approximately two-thirds of reviewed patients infected or colonised by fluconazole-resistant C. tropicalis were azole-naïve. Furthermore, there was no evidence of patient-to-patient transmission. Because the clade 4 fluconazole-resistant C. tropicalis strain persists in Taiwan, it is important to identify the source of azole-resistant C. tropicalis to prevent the spread of this resistant strain.

The nonstructural antigen protein 3 of the hepatitis C virus (HCV NS3), commonly-used for HCV ELISA diagnosis, possesses protease and helicase activities. To prevent auto-degradation, a truncated NS3 protein was designed by removing the protease domain. Firstly, it was overexpressed in E. coli by IPTG induction under two different temperatures (25 and 37 °C), and purified using affinity chromatography to attain homogeneity above 90%. The molecular mass of purified protein was determined to be approx. 55 kDa. While lowering the temperature from 37 to 25 °C, the yield of the soluble fraction of HCV NS3 was increased from 4.15 to 11.1 mgL−1 culture, which also improved the antigenic activity and specificity. The protein stability was investigated after long-term storage (for 6 months at −20 °C) revealed no loss of activity, specificity, or antigenic efficacy. A thermal stability study on both freshly produced and stored HCV NS3 fractions at both temperatures showed that the unfolding curve profile properly obey the three-state unfolding mechanism. In the first transition phase, the midpoints of the thermal denaturation of fresh NS3 produced at 37 °C and 25 °C, and that produced after long-term storage at 37 °C and 25 °C, were 59.7 °C, 59.1 °C, 55.5 °C, and 57.8 °C, respectively. Microplates coated with the fresh NS3 produced at 25 °C or at 37 °C that were used for the HCV ELISA test and the diagnosis outcome were compared with two commercial kits—Abbott HCV EIA 2.0 and Ortho HCV EIA 3.0. Results indicated that the specificity of the HCV NS3 produced fresh at 25 °C was higher than that of the fresh one at 37 °C, hence showing potential for application in HCV ELISA diagnosis.

Background Human gut microbiome has an essential role in human health and disease. Although the major dominant microbiota within individuals have been reported, the change of gut microbiome caused by external factors, such as antibiotic use and bowel cleansing, remains unclear. We conducted this study to investigate the change of gut microbiome in overweight male adults after bowel preparation, where none of the participants had been diagnosed with any systemic diseases. Methods A total of 20 overweight, male Taiwanese adults were recruited, and all participants were omnivorous. The participants provided fecal samples and blood samples at three time points: prior to bowel preparation, 7 days after colonoscopy, and 28 days after colonoscopy. The microbiota composition in fecal samples was analyzed using 16S ribosome RNA gene amplicon sequencing. Results Our results demonstrated that the relative abundance of the most dominant bacteria hardly changed from prior to bowel preparation to 28 days after colonoscopy. Using the ratio of Prevotella to the sum of Prevotella and Bacteroides in the fecal samples at baseline, the participants were separated into two groups. The fecal samples of the Type 1 group was Bacteroides-dominant, and that of the Type 2 group was Prevotella-dominant with a noticeable presence Bacteroides. Bulleidia appears more in the Type 1 fecal samples, while Akkermensia appears more in the Type 2 fecal samples. Of each type, the gut microbial diversity differed slightly among the three collection times. Additionally, the Type 2 fecal microbiota was temporarily susceptible to bowel cleansing. Predictive functional analysis of microbial community reveals that their activities for the mineral absorption metabolism and arachidonic acid metabolism differed significantly between the two types. Depending on their fecal type, the variance of triglycerides and C-reactive protein also differed between the two types of participants. Conclusions Depending upon the fecal type, the microbial diversity and the predictive functional modules of microbial community differed significantly after bowel preparation. In addition, blood biochemical markers presented somewhat associated with fecal type. Therefore, our results might provide some insights as to how knowledge of the microbial community could be used to promote health through personalized clinical treatment. Electronic supplementary material The online version of this article (10.1186/s12864-018-5285-6) contains supplementary material, which is available to authorized users.

In this work, a new analytical approach combining optical microscope with software was carried out to study the effect of music on bacteria and to explore its potential mechanism. The stimulation of classical music (Flight of the Bumblebee) with different frequencies (range from 55 Hz to 4186 Hz) and tempos (range from 25 BPM to 250 BPM) was transmitted in the form of vibration by the apparatus which was bone conduction to Escherichia coli MG1655. The increment of E. coli motility affected by Flight of the Bumblebee was quantified by two approaches. One was indirect motility assay and the other was the in-situ monitoring on the swimming behavior of E. coli under the optical microscope. The in-situ monitoring was conducted by combining optical microscope with software which enabled real-time observation on the swimming behavior of E. coli. Both results showed that different frequencies and tempos of musical vibration affected the motility to a different extent. With comparison, it was found that the music with higher frequency and faster tempo could enhance more on the motility. Above findings suggest this new analytical approach could be applied for investigating the motility of E. coli, and also a potential mechanism to modulate biological cells for future digital therapeutic and medical device applications.

Figure S7. Box plots of three inflammation cytokines. (PDF 377â kb)

Figure S2. Principal coordinate analysis (PCoA) based on the UniFrac distance values. (PDF 414â kb)

Figsure S3-S5. Correlation networks for 61 genera at three sample collection times. (PDF 2498â kb)

Table S10. Means of 26 predictive functional modules between the Type 1 and Type 2 fecal samples. (PDF 209â kb)

Figure S1. The differences of microbial diversity between the type 1 and 2 groups. (PDF 331â kb)

Figure S6. Percentage of the number of correlation change between SB and S7; SB and S28. (PDF 232â kb)

Table S3. Descriptive statistics of richness and Shannon diversity index. (PDF 184â kb)

This paper presents the performance of Pt deposited by different deposition methods, including electrodeposition, e-gun, and sputtering on a carbon cloth as a cathode in a microbial fuel cell (MFC) system. The results indicate that Pt deposited by electrodeposition exhibits the highest active efficiency on hydrogen electroabsorption. According to XPS fitting results, the amount of Pt (0), zero-valent Pt, existing in Pt deposited on carbon cloth by electrodeposition was larger than that in Pt deposited by e-gun and sputtering. The cyclic voltammogram analyses also show more real active surface area of Pt deposited by electrodeposition, mostly contributed by its smaller particle size/shape and higher catalytic effect. Besides, the total impedance of Pt deposited by electrodeposition was lower than that of Pt deposited by e-gun and sputtering from EIS measurement at 0.1 Hz. Applied to MFC system, the highest power density was manifested and the least Pt loading were observed for the Pt deposited on carbon cloth by electrodeposition. It is therefore can be concluded that the electrodeposited-Pt exhibit superior performance as a cathode for future MFC applications.

Klebsiella pneumoniae CG43, a heavy encapsulated liver abscess isolate, mainly expresses type 3 fimbriae. Type 1 fimbriae expression was only apparent in CG43S3ΔmrkA (the type 3 fimbriae-deficient strain). The expression of type 1 fimbriae in CG43S3ΔmrkA was reduced by deleting the fimK gene, but was unaffected by removing the 3' end of fimK encoding the C-terminal EIL domain (EILfimK). Quantitative RT-PCR and promoter activity analysis showed that the putative DNA-binding region at the N terminus, but not the C-terminal EIL domain, of FimK positively affects transcription of the type 1 fimbrial major subunit, fimA. An electrophoretic mobility shift assay demonstrated that the recombinant FimK could specifically bind to fimS, which is located upstream of fimA and contains a vegetative promoter for the fim operon, also reflecting possible transcriptional regulation. EILfimK was shown to encode a functional phosphodiesterase (PDE) via enhancing motility in Escherichia coli JM109 and in vitro using PDE activity assays. Moreover, EILfimK exhibited higher PDE activity than FimK, implying that the N-terminal DNA-binding domain may negatively affect the PDE activity of FimK. FimA expression was detected in CG43S3 expressing EILfimK or AILfimK, suggesting that FimA expression is not directly influenced by the c-di-GMP level. In summary, FimK influences type 1 fimbriation by binding to fimS at the N-terminal domain, and thereafter, the altered protein structure may activate C-terminal PDE activity to reduce the intracellular c-di-GMP level.

Klebsiella pneumoniae is the predominant pathogen isolated from liver abscesses of diabetic patients in Asian countries. However, the effects of elevated blood glucose levels on the virulence of this pathogen remain largely unknown. Type 3 fimbriae, encoded by the mrkABCDF genes, are important virulence factors in K. pneumoniae pathogenesis. In this study, the effects of exogenous glucose and the intracellular cyclic AMP (cAMP) signaling pathway on type 3 fimbriae expression regulation were investigated. The production of MrkA, the major subunit of type 3 fimbriae, was increased in glucose-rich medium, whereas cAMP supplementation reversed the effect. MrkA production was markedly increased by cyaA or crp deletion, but slightly decreased by cpdA deletion. In addition, the mRNA levels of mrkABCDF genes and the activity of PmrkA were increased in Δcrp strain, as well as the mRNA levels of mrkHIJ genes that encode cyclic di-GMP (c-di-GMP)-related regulatory proteins that influence type 3 fimbriae expression. Moreover, the activities of PmrkHI and PmrkJ were decreased in ΔlacZΔcrp strain. These results indicate that CRP-cAMP down-regulates mrkABCDF and mrkHIJ at the transcriptional level. Further deletion of mrkH or mrkI in Δcrp strain diminished the production of MrkA, indicating that MrkH and MrkI are required for the CRP regulation of type 3 fimbriae expression. Furthermore, the high activity of PmrkHI in the ΔlacZΔcrp strain was diminished in ΔlacZΔcrpΔmrkHI, but increased in the ΔlacZΔcrpΔmrkJ strain. Deletion of crp increased the intracellular c-di-GMP concentration and reduced the phosphodiesterase activity. Moreover, we found that the mRNA levels of multiple genes related to c-di-GMP metabolism were altered in Δcrp strain. These indicate that CRP regulates type 3 fimbriae expression indirectly via the c-di-GMP signaling pathway. In conclusion, we found evidence of a coordinated regulation of type 3 fimbriae expression by the CRP-cAMP and c-di-GMP signaling pathways in K. pneumoniae.

This study investigated the structural and mechanical properties of Klebsiella pneumoniae type 3 fimbriae, which constitute a known virulence factor for the bacterium. Transmission electron microscopy and optical tweezers were used to understand the ability of the bacterium to survive flushes. An individual K. pneumoniae type 3 fimbria exhibited a helix-like structure with a pitch of 4.1 nm and a three-phase force-extension curve. The fimbria was first nonlinearly stretched with increasing force. Then, it started to uncoil and extended several micrometers at a fixed force of 66 ± 4 pN ( n = 22). Finally, the extension of the fimbria shifted to the third phase, with a characteristic force of 102 ± 9 pN ( n = 14) at the inflection point. Compared with the P fimbriae and type 1 fimbriae of uropathogenic Escherichia coli , K. pneumoniae type 3 fimbriae have a larger pitch in the helix-like structure and stronger uncoiling and characteristic forces.

The ferric uptake regulator Fur has been reported to repress the expression ofrmpA, a regulatory gene for the mucoid phenotype, leading to decreased capsular polysaccharide (CPS) biosynthesis inKlebsiella pneumoniaeCG43. Here, quantitative real-time PCR (qRT-PCR) analyses and electrophoretic mobility shift assays showed that Fur also repressed the expression of the CPS regulatory genesrmpA2andrcsA. Interestingly, deletion ofrmpAorrcsAbut notrmpA2from the Δfurstrain was able to suppress the deletion effect of Fur. The availability of extracellular iron affected the amount of CPS, suggesting that Fur regulates CPS biosynthesis in an Fe(II)-dependent manner. Increased production of siderophores was observed in the Δfurstrain, suggesting that uptake of extracellular iron inK. pneumoniaeis regulated by Fur. Fur titration assays and qRT-PCR analyses demonstrated that at least six of the eight putative iron-acquisition systems, identified by ablastsearch in the contig database ofK. pneumoniaeCG43, were directly repressed by Fur. We conclude that Fur has a dual role in the regulation of CPS biosynthesis and iron acquisition inK. pneumoniae.

In order to enhance the sensitivity of diagnosis, a recombinant clone containing domain I of HCV core (amino acid residues 1 to 123) was subjected to random mutagenesis. Five mutants with higher sensitivity were obtained by colony screening of 616 mutants using reverse ELISA. Sequence analysis of these mutants revealed alterations focusing on W84, P95, P110, or V129. The inclusion bodies of these recombinant proteins overexpressed inE. coliBL21(DE3) were subsequently dissolved using 6 M urea and then refolded by stepwise dialysis. Compared to the unfolded wild-type antigen, the refolded M3b antigen (W84S, P110S and V129L) exhibited an increase of 66% antigenicity with binding capacity of 0.96 and affinity of 113 μM−1. Moreover, the 33% decrease of the production demand suggests that M3b is a potential substitute for anti-HCV antibody detection.

Sequence analysis of the large virulence plasmid pLVPK inKlebsiella pneumoniaeCG43 revealed the presence of another mucoid factor encoding genermpAbesidesrmpA2. Promoter activity measurement indicated that the deletion ofrmpAreduced K2 capsular polysaccharide (CPS) biosynthesis, resulting in decreased colony mucoidy and virulence in mice. Introduction of a multicopy plasmid carryingrmpArestored CPS production in thermpAorrmpA2mutant but not in thercsBmutant. Transformation of thermpAdeletion mutant with anrcsB-carrying plasmid also failed to enhance CPS production, suggesting that a cooperation of RmpA with RcsB is required for regulatory activity. This was further corroborated by the demonstration ofin vivointeraction between RmpA and RcsB using two-hybrid analysis and coimmunoprecipitation analysis. A putative Fur binding box was only found at the 5′ noncoding region ofrmpA. The promoter activity analysis indicated that the deletion offurincreased thermpApromoter activity. Using electrophoretic mobility shift assay, we further demonstrated that Fur exerts its regulatory activity by binding directly to the promoter. As a result, thefurdeletion mutant exhibited an increase in colony mucoidy, CPS production, and virulence in mice. In summary, our results suggested that RmpA activates CPS biosynthesis inK. pneumoniaeCG43 via an RcsB-dependent manner. The expression ofrmpAis regulated by the availability of iron and is negatively controlled by Fur.

Klebsiella pneumoniae is the predominant pathogen of primary liver abscess. However, our knowledge regarding the molecular basis of how K. pneumoniae causes primary infection in the liver is limited. We established an oral infection model that recapitulated the characteristics of liver abscess and conducted a genetic screen to identify the K. pneumoniae genes required for the development of liver abscess in mice. Twenty-eight mutants with attenuated growth in liver or spleen samples out of 2,880 signature-tagged mutants that produced the wild-type capsule were identified, and genetic loci which were disrupted in these mutants were identified to encode products with roles in cellular metabolism, adhesion, transportation, gene regulation, and unknown functions. We further evaluated the virulence attenuation of these mutants in independent infection experiments and categorized them accordingly into three classes. In particular, the class I and II mutant strains exhibited significantly reduced virulence in mice, and most of these strains were not detected in extraintestinal tissues at 48 h after oral inoculation. Interestingly, the mutated loci of about one-third of the class I and II mutant strains encode proteins with regulatory functions, and the transcript abundances of many other genes identified in the same screen were markedly changed in these regulatory mutant strains, suggesting a requirement for genetic regulatory networks for translocation of K. pneumoniae across the intestinal barrier. Furthermore, our finding that preimmunization with certain class I mutant strains protected mice against challenge with the wild-type strain implied a potential application for these strains in prophylaxis against K. pneumoniae infections.

Vibrio vulnificus YJ016 contains three genes encoding proteins homologous to repeats-in-toxin proteins. One of these genes, vva0331, possesses a long open reading frame of 13,971 bp in length and resides on the small chromosome between two gene clusters encoding a type I secretion system and several regulatory proteins, respectively. Bioinformatic analysis revealed that VVA0331 consist of nineteen 87-amino acid repeats, two Arg-Gly-Asp motifs, four cysteine residues, an outer membrane protein domain, a polysaccharide-binding site and several motifs related to cell adhesions. These features are distinct from those of typical repeat-in-toxins and autotransporter adhesins. Real-time quantitative PCR analysis indicates that vva0331 gene expression is activated at 30 degrees C and regulated by iron. In addition, VVA0331 is present primarily in a secreted form as determined by cell fractionation assay and Western blot analysis. No significant difference in Hep2 cell adherence, cytotoxicity, and virulence was observed between the wild type and vva0331 mutant strains. In contrast, these strains exhibited apparently different outer membrane protein profiles, and antiserum raised against C-terminal region of VVA0331 reacted with an 85-kDa outer membrane protein of V. vulnificus YJ016.

Many bacterial species utilize acetoin as a carbon source. In Klebsiella pneumoniae, the utilization of acetoin is catalyzed by an acetoin dehydrogenase complex encoded by the acoABCD operon, which is positively regulated in the presence of acetoin by the transcriptional factor AcoK. AcoK contains a LuxR type DNA-binding domain at the C-terminal region and putative Walker A and B nucleotide-binding motifs in the N-terminal region. The comprehensive deletion and mutation study performed here shows that mutations in the putative Walker A motif result in a significant reduction of ATP hydrolysis and trans-activation by AcoK of acoABCD expression, presumably due to a loss of ATP-binding ability. AcoK was shown to bind specifically to nucleotides -66 to -36 of the acoABCD promoter, though the DNA-binding ability was not affected by the Walker A motif mutation. Thus, this study provides an additional example of how a member of the signal transduction ATPases with numerous domains family activates its target gene expression.

The mechanism of DNA transfer from Escherichia coli ( E. coli) Hfr donor strain AT2453 to recipient strain AB1157 during the conjugation process has been investigated by liquid atomic force microscopy (AFM). With the success of immobilizing both E. coli strains on gelatin-treated glass under aqueous solution, the F-pilus between an E. coli mating pair could be clearly imaged and dissected by an AFM probe. Another AFM probe functionalized with an anti-single-stranded DNA (ssDNA) antibody was then applied to detect transferring ssDNA. According to the AFM force spectrum, the transferring ssDNA could be detected only in the dissected area with a binding force of 109 +/- 5 pN measured. Our results provide direct evidence indicating that the DNA was transferred through the F-pilus channel between an E. coli mating pair during their conjugation.

Certain bacterial two-component sensor kinases possess a histidine-containing phosphotransfer (Hpt) domain to carry out a multistep phosphotransferring reaction to a cognate response regulator. Pseudomonas aeruginosa PAO1 contains three genes that encode proteins with an Hpt domain but lack a kinase domain. To identify the sensor kinase coupled to these Hpt proteins, a phosphorelay profiling assay was performed. Among the 12 recombinant orphan sensor kinases tested, 4 of these sensors (PA1611, PA1976, PA2824, and RetS) transferred the phosphoryl group to HptB (PA3345). The in vivo interaction between HptB and each of the sensors was also confirmed using the bacterial two-hybrid assay. Interestingly, the phosphoryl groups from these sensors all appeared to be transferred via HptB to PA3346, a novel phosphatase consisting of an N-terminal receiver domain and a eukaryotic type Ser/Thr phosphatase domain, and resulted in a significant increase of its phosphatase activity. The subsequent reverse transcription-PCR analysis revealed an operon structure of hptB-PA3346–PA3347, suggesting a coordinate expression of the three genes to carry out a signal transduction. The possibility was supported by the analysis showing PA3347 is able to be phosphorylated on Ser-56, and this phosphoryl group could be removed by PA3346 protein. Finally, analysis of PA3346 and PA3347 gene knock-out mutants revealed that these genes are associated with bacterial swarming activity and biofilm formation. Together, these results disclose a novel multistep phosphorelay system that is essential for P. aeruginosa to respond to a wide spectrum of environmental signals.

PmrA, an OmpR/PhoB family response regulator, manages genes for antibiotic resistance. Phosphorylation of OmpR/PhoB response regulator induces the formation of a symmetric dimer in the N-terminal receiver domain (REC), promoting two C-terminal DNA-binding domains (DBDs) to recognize promoter DNA to elicit adaptive responses. Recently, determination of the KdpE–DNA complex structure revealed an REC–DBD interface in the upstream protomer that may be necessary for transcription activation. Here, we report the 3.2-Å-resolution crystal structure of the PmrA–DNA complex, which reveals a similar yet different REC–DBD interface. However, NMR studies show that in the DNA-bound state, two domains tumble separately and an REC–DBD interaction is transiently populated in solution. Reporter gene analyses of PmrA variants with altered interface residues suggest that the interface is not crucial for supporting gene expression. We propose that REC–DBD interdomain dynamics and the DBD–DBD interface help PmrA interact with RNA polymerase holoenzyme to activate downstream gene transcription., PmrA is a regulator of genes that are associated with antibiotic resistance. Here, the authors report the structure of the protein bound to DNA, and use NMR data to propose a mechanism for the regulation of gene transcription.

In the Klebsiella pneumoniae CG43 genome, the divergently transcribed genes coding for PecS, the MarR-type transcription factor, and PecM, the drug metabolite transporter, are located between the type 1 and type 3 fimbrial gene clusters. The intergenic sequence pecO between pecS and pecM contains three putative PecS binding sites and a CpxR box. Electrophoretic mobility shift assay revealed that the recombinant PecS and CpxR could specifically bind to the pecO sequence, and the specific interaction of PecS and pecO could be attenuated by urate. The expression of pecS and pecM was negatively regulated by CpxAR and PecS, and was inducible by exogenous urate in the absence of cpxAR. Compared with CG43S3ΔcpxAR, the derived mutants CG43S3ΔcpxARΔpecS and CG43S3ΔcpxARΔpecSΔpecM exerted similar levels of sensitivity to H2O2 or paraquat, but higher levels of mannose-sensitive yeast agglutination activity and FimA production. The promoter activity and transcript levels of fimA in CG43S3ΔcpxAR were also increased by deleting pecS. However, no binding activity between PecS and the fimA promoter could be observed. Nevertheless, PecS deletion could reduce the expression of the global regulator HNS and release the negative effect of HNS on FimA expression. In CG43S3ΔcpxAR, the expression of FimA as well as PecS was inducible by urate, whilst urate-induced FimA expression was inhibited by the deletion of pecS. Taken together, we propose that K. pneumoniae PecS indirectly and negatively regulates the expression of type 1 fimbriae, and the regulation is urate-inducible in the absence of CpxAR.

Type 3 fimbriae are important adhesive filaments that assist Klebsiella pneumoniae to establish an infection. Different MrkD adhesin variants on the fimbriae are known to display distinct adherence capability for the bacteria to bind extracellular matrix proteins, although the difference has not been determined physically. For this reason, the adhesive force between type 3 fimbriae and collagen IV were measured using optical tweezers. The measured force data displayed a periodic histogram thus Fourier analysis was applied to group it to extract the adhesive force of a single molecular pair. Specifically, we showed that grouping should begin with an offset at the first half of the period. Finally, we first present the adhesive force between each mrkD(V2)-, mrkD(V3)-, and mrkD(V4)-expressed fimbriae and collagen IV is 2.03, 3.79, and 2.87 pN, respectively. This result can be referred to further research on mrkD allelic effect on bacteria infection.

In Klebsiella pneumoniae CG43, deletion of the sensor gene kvgS reduced the kvgAS expression in M9 medium with 0.2 mM paraquat, 0.2 mM 2,2-dihydropyridyl, or 300 mM NaCl. This result shows an autoregulatory role of KvgS and a stress-responsive expression of the two-component system (2CS). The kvgS deletion also appeared to decrease the expression of kvhAS, paralogous genes of kvgAS. Additionally, measurements of the promoter activity in kvgA(-) mutant revealed that KvgA is probably an activator for the expression of kvgAS and kvhAS. The subsequent electrophoretic mobility shift assay, indicating a specific binding of the recombinant KvgA to the putative promoters P(kvgAS) and P(kvhAS), also supported an interacting regulation between the 2CSs. In P(kvgAS) and P(kvhAS), the presence of RpoS binding elements suggested an RpoS-dependent regulation. Nevertheless, the rpoS deletion reduced the expression of kvgAS but increased that of kvhAS. Moreover, the kvgA deletion reduced the expression of katG and sodC. The overexpression of KvhA altered the susceptibility to fosfomycin and an increasing activity of UDP-N-acetylglucosamine enolpyruvyl transferase, the target protein of fosfomycin, which suggesting a regulation by KvhA. Taken together, these indicated that the two 2CSs probably belong to different regulatory circuits of the RpoS regulon.

On the basis of phenotypic analysis, the Klebsiella pneumoniae CG43 derived mutants with deletions of the gene encoding respectively the response regulators KvgA, KvhA, and KvhR were classified into two groups. Group I bacteria carrying either kvgA - or kvhR - exhibited less mucoidy, lower level of capsular polysaccharide (CPS) synthesis and higher LD50 than the parental strain. No apparent change of the group II, including kvhA - and kvhA - kvhR - mutants, was observed. However, the mucoidy of kvhA - kvhR mutant was found to be diminished after introducing into a kvhA - expressing plasmid. Via promoter-lacZ fusion analysis, kvhA deletion was found to reduce kvhR expression. A regulatory role of KvhA for the expression of kvhR was supported further by EMSA showing a specific binding of KvhA to the putative promoter of kvhR. The promoter activity measurement and EMSA also revealed that KvgA acted as an autoregulator and an activator for the expression of kvhAS and kvhR. In addition, deletion of kvgA suppressed slightly the promoter activity of the cps-orf16-17, and the expression of all three cps transcripts orf1-2, orf3-15, and orf16-17 were reduced in the kvhR - mutant. These suggest that the three homologous response regulators interact to control, in coordination, the bacterial cps expression.

We herein demonstrate that the hybrid sensor PA1611 carries out specific signal transduction, through HptB (PA3345), to the response regulator PA3346 in Pseudomonas aeruginosa PAO1. As assessed by phenotypic changes in the hptB deletion mutant, the pathway is likely to be involved in the regulation of flagellar activity, the chemotaxis response, twitching motility, and biofilm formation in the bacteria.

The enzyme UDP-glucose dehydrogenase (UGDH) catalyzes the conversion of UDP-glucose to UDP-glucuronic acid, which is essential for the biosynthesis of complex carbohydrates such as hyaluronan in many cell types, and is required for detoxification of toxic compounds in the liver. We previously defined the 714 bp 5'-flanking region of the UGDH gene as the core promoter, with putative negative regulatory elements residing in the region upstream of it. In the present study, we delineated the region from nucleotide positions -1057 to -957 on the UGDH promoter to be responsible for the repression of promoter activity. A mutation at nucleotide -1003, which is contained within a motif predicted to be the response element for peroxisome proliferator receptor alpha (PPARalpha), abolished the suppression effect. DNA-protein interaction was observed at this motif by electrophoretic mobility shift assay. The proteins interacting with the PPRE-like repressor motif were purified by biotin-labeled DNA affinity chromatography. Subsequently, MALDI-TOF identified the purified proteins as a 62-kDa zinc finger and a 42-kDa beta-actin protein. Hence in this study we report the presence of an inhibitory cis-element in the distal region of the UGDH promoter that interacts with putative transcriptional repressors for the negative regulation of the UGDH gene.

We have determined the entire DNA sequence of pLVPK, which is a 219-kb virulence plasmid harbored in a bacteremic isolate of Klebsiella pneumoniae. A total of 251 open reading frames (ORFs) were annotated, of which 37% have homologous genes of known function, 31% match the hypothetical genes in the GenBank database, and the remaining 32% are novel sequences. The obvious virulence-associated genes carried by the plasmid are the capsular polysaccharide synthesis regulator rmpA and its homolog rmpA2, and multiple iron-acquisition systems, including iucABCDiutA and iroBCDN siderophore gene clusters, Mesorhizobium loti fepBC ABC-type transporter, and Escherichia coli fecIRA, which encodes a Fur-dependent regulatory system for iron uptake. In addition, several gene clusters homologous with copper, silver, lead, and tellurite resistance genes of other bacteria were also identified. Identification of a replication origin consisting of a repA gene lying in between two sets of iterons suggests that the replication of pLVPK is iteron-controlled and the iterons are the binding sites for the repA to initiate replication and maintain copy number of the plasmid. Genes homologous with E. coli sopA/sopB and parA/parB with nearby direct DNA repeats were also identified indicating the presence of an F plasmid-like partitioning system. Finally, the presence of 13 insertion sequences located mostly at the boundaries of the aforementioned gene clusters suggests that pLVPK was derived from a sequential assembly of various horizontally acquired DNA fragments.

The rmpA2 gene, which encodes an activator for capsular polysaccharide (CPS) synthesis, was isolated from a 200-kb virulence plasmid of Klebsiella pneumoniae CG43. Based on the sequence homology with LuxR at the carboxyl-terminal DNA-binding motif, we hypothesized that RmpA2 exerts its effect by activating the expression of cps genes that are responsible for CPS biosynthesis. Two luxAB transcriptional fusions, each containing a putative promoter region of the K. pneumoniae K2 cps genes, were constructed and were found to be activated in the presence of multicopy rmpA2 . The activation is likely due to direct binding of RmpA2 to the cps gene promoter through its C-terminal DNA binding motif. Moreover, the loss of colony mucoidy in a K. pneumoniae strain deficient in RcsB, a regulator for cps gene expression, could be recovered by complementing the strain with a multicopy plasmid carrying rmpA2 . The CPS production in Lon protease-deficient K. pneumoniae significantly increased, and the effect was accompanied by an increase of RmpA2 stability. The expression of the rmpA2 gene was negatively autoregulated and could be activated when the organism was grown in M9 minimal medium. An IS 3 element located upstream of the rmpA2 was required for the full activation of the rmpA2 promoter. In summary, our results suggest that the enhancement of K2 CPS synthesis in K. pneumoniae CG43 by RmpA2 can be attributed to its transcriptional activation of K2 cps genes, and the expression level of rmpA2 is autoregulated and under the control of Lon protease.

A two-component system encoding gene cluster kvgAS that is present only in virulent Klebsiella pneumoniae CG43 was isolated and its sequence determined. RT-PCR and Southern analysis demonstrated that kvgAS is organized as an operon. No apparent effect of a kvgS deletion on bacterial virulence was observed in a mouse peritonitis model. In the presence of paraquat or 2,2-dipyridyl, the activity of kvgAS promoter in the kvgS mutant was found to be reduced to half of the level in the wild-type strain. The data suggest that the KvgAS system is autoregulated and plays a role in countering free radical stresses and sensing iron-limiting conditions.

The Klebsiella pneumoniae mdcR gene, which encodes a LysR-type regulator, was overexpressed in Escherichia coli . Purified MdcR was found to bind specifically to the control region of either the malonate decarboxylase ( mdc ) genes or mdcR . We have also demonstrated that MdcR is an activator of the expression of the mdc genes, whereas it represses the transcription of the putative control region of mdcR , P mdcR , indicating a negative autoregulatory control.

During a 4-month period, 9 premature infants hospitalized in a neonatal intensive care unit (NICU) developed Candida albicans fungaemia. All 9 infants received antifungal agents. Fluconazole was administered in 7 patients and successfully eradicated this organism in 6 with no adverse effects. For epidemiological investigation, 64 environmental specimens and hand-washings of all 54 staff members involved in the NICU were examined for the presence of this organism. No C. albicans could be identified from environmental sources, while the hand-washing of 1 nurse was C. albicans-positive. Two genotyping methods, including electrophoretic karyotyping using contour-clamped homogeneous electric field gel electrophoresis and polymerase chain reaction-based direct sequencing of rRNA gene, were used in the analysis of the isolates recovered from blood cultures of the infants, the hand-washing of the nurse and 7 control isolates. Both methods yielded comparable results and revealed that all 13 isolates from infected infants as well as the isolate from hand washing of the nurse were of the same genotype while the control isolates were distinct. These results suggest that the outbreak of C. albicans fungaemia was caused by a particular strain and possibly via cross-infection. In addition, we showed that fluconazole seemed to be safe and effective in treating C. albicans fungaemia in neonates, although the data were limited.

By using transposon insertional mutagenesis and deletion analyses, a recombinant clone containing the region upstream of the acoABCD operon of Klebsiella pneumoniae was found to be required for acetoin-inducible expression of the operon in Escherichia coli. The nucleotide sequence of the region was determined, and it displayed an open reading frame of 2,763 bp that is transcribed divergently to the acoABCD operon. This gene, designated acoK, is capable of encoding a protein with an overall 58.4% amino acid identity with MalT, the transcriptional activator of the E. coli maltose regulon. A conserved sequence for nucleotide binding at the N-terminal region, as well as a helix-turn-helix motif belonging to the LuxR family of transcriptional regulators at the C terminus, was also identified. Primer extension analysis identified two transcription initiation sites, S1 and S2, located 319 and 267 bp, respectively, upstream of the putative start codon of acoK. Several copies of NtrC recognition sequence [CAC-(N11 to N18)-GTG] were found in the promoter regions of both the acoK gene and the acoABCD operon. Acetoin-dependent expression of the acoABCD operon could be restored in the E. coli acoK mutants by supplying a plasmid carrying an intact acoK, suggesting a transactivating function of the gene product. The AcoK protein overproduced in E. coli was approximately 100 kDa, which is in good agreement with the molecular mass deduced from the nucleotide sequence. A specific DNA binding property and an ATPase activity of the purified AcoK were also demonstrated.

A lobule-mimetic cell-patterning technique for on-chip reconstruction of centimetre-scale liver tissue of heterogeneous hepatic and endothelial cells via an enhanced field-induced dielectrophoresis (DEP) trap is demonstrated and reported. By mimicking the basic morphology of liver tissue, the classic hepatic lobule, the lobule-mimetic-stellate-electrodes array was designed for cell patterning. Through DEP manipulation, well-defined and enhanced spatial electric field gradients were created for in-parallel manipulation of massive individual cells. With this liver-cell patterning labchip design, the original randomly distributed hepatic and endothelial cells inside the microfluidic chamber can be manipulated separately and aligned into the desired pattern that mimicks the morphology of liver lobule tissue. Experimental results showed that both hepatic and endothelial cells were orderly guided, snared, and aligned along the field-induced orientation to form the lobule-mimetic pattern. About 95% cell viability of hepatic and endothelial cells was also observed after cell-patterning demonstration via a fluorescent assay technique. The liver function of CYP450-1A1 enzyme activity showed an 80% enhancement for our engineered liver tissue (HepG2+HUVECs) compared to the non-patterned pure HepG2 for two-day culturing.

This study shows that the expression of yjcC, an in vivo expression (IVE) gene, and the stress response regulatory genes soxR, soxS, and rpoS are paraquat inducible in Klebsiella pneumoniae CG43. The deletion of rpoS or soxRS decreased yjcC expression, implying an RpoS- or SoxRS-dependent control. After paraquat or H2O2 treatment, the deletion of yjcC reduced bacterial survival. These effects could be complemented by introducing the ΔyjcC mutant with the YjcC-expression plasmid pJR1. The recombinant protein containing only the YjcC-EAL domain exhibited phosphodiesterase (PDE) activity; overexpression of yjcC has lower levels of cyclic di-GMP. The yjcC deletion mutant also exhibited increased reactive oxygen species (ROS) formation, oxidation damage, and oxidative stress scavenging activity. In addition, the yjcC deletion reduced capsular polysaccharide production in the bacteria, but increased the LD50 in mice, biofilm formation, and type 3 fimbriae major pilin MrkA production. Finally, a comparative transcriptome analysis showed 34 upregulated and 29 downregulated genes with the increased production of YjcC. The activated gene products include glutaredoxin I, thioredoxin, heat shock proteins, chaperone, and MrkHI, and proteins for energy metabolism (transporters, cell surface structure, and transcriptional regulation). In conclusion, the results of this study suggest that YjcC positively regulates the oxidative stress response and mouse virulence but negatively affects the biofilm formation and type 3 fimbriae expression by altering the c-di-GMP levels after receiving oxidative stress signaling inputs.

Background The opportunistic enterobacterium, Morganella morganii, which can cause bacteraemia, is the ninth most prevalent cause of clinical infections in patients at Changhua Christian Hospital, Taiwan. The KT strain of M. morganii was isolated during postoperative care of a cancer patient with a gallbladder stone who developed sepsis caused by bacteraemia. M. morganii is sometimes encountered in nosocomial settings and has been causally linked to catheter-associated bacteriuria, complex infections of the urinary and/or hepatobiliary tracts, wound infection, and septicaemia. M. morganii infection is associated with a high mortality rate, although most patients respond well to appropriate antibiotic therapy. To obtain insights into the genome biology of M. morganii and the mechanisms underlying its pathogenicity, we used Illumina technology to sequence the genome of the KT strain and compared its sequence with the genome sequences of related bacteria. Results The 3,826,919-bp sequence contained in 58 contigs has a GC content of 51.15% and includes 3,565 protein-coding sequences, 72 tRNA genes, and 10 rRNA genes. The pathogenicity-related genes encode determinants of drug resistance, fimbrial adhesins, an IgA protease, haemolysins, ureases, and insecticidal and apoptotic toxins as well as proteins found in flagellae, the iron acquisition system, a type-3 secretion system (T3SS), and several two-component systems. Comparison with 14 genome sequences from other members of Enterobacteriaceae revealed different degrees of similarity to several systems found in M. morganii. The most striking similarities were found in the IS4 family of transposases, insecticidal toxins, T3SS components, and proteins required for ethanolamine use (eut operon) and cobalamin (vitamin B12) biosynthesis. The eut operon and the gene cluster for cobalamin biosynthesis are not present in the other Proteeae genomes analysed. Moreover, organisation of the 19 genes of the eut operon differs from that found in the other non-Proteeae enterobacterial genomes. Conclusions This is the first genome sequence of M. morganii, which is a clinically relevant pathogen. Comparative genome analysis revealed several pathogenicity-related genes and novel genes not found in the genomes of other members of Proteeae. Thus, the genome sequence of M. morganii provides important information concerning virulence and determinants of fitness in this pathogen.

Comparison of the amino acid sequences of five eukaryotic UDPglucose pyrophosphorylases has identified a number of conserved residues that may be important for substrate binding or catalysis. Using the cloned cDNA for the human liver enzyme, we have investigated the role of several of these residues by site-directed mutagenesis. Changing the single conserved cysteine (residue 123) to serine resulted in an active enzyme, as did mutating the single conserved histidine (residue 266) to arginine. The two conserved tryptophans were each altered to serine; W218S is active while W333S is not. In the latter case, the enzyme does not appear to fold correctly, and a similar result was obtained by mutation to lysine at one (residue 391) of the four conserved arginines. The other three arginines are not essential, as judged by the observation that R389H, R422Q and R445H are all active. The kinetic properties of each active mutant were investigated and in most cases were found to be similar to those of wild-type. The most dramatic change is a sevenfold increase in the K(m) for magnesium pyrophosphate with C123S. Overall, none of these conserved residues appears to be essential for activity, although such a role cannot be ruled out for W333 and R391 where mutation resulted in defective folding.

The Klebsiella pneumoniae type 3 fimbriae are mainly composed of MrkA pilins that assemble into a helixlike filament. This study determined the biomechanical properties of the fimbriae and analyzed 11 site-directed MrkA mutants to identify domains that are critical for the properties. Escherichia coli strains expressing type 3 fimbriae with an Ala substitution at either F34, V45, C87, G189, T196, or Y197 resulted in a significant reduction in biofilm formation. The E. coli strain expressing MrkAG189A remained capable of producing a normal number of fimbriae. Although F34A, V45A, T196A, and Y197A substitutions expressed on E. coli strains produced sparse quantities of fimbriae, no fimbriae were observed on the cells expressing MrkAC87A. Further investigations of the mechanical properties of the MrkAG189A fimbriae with optical tweezers revealed that, unlike the wild-type fimbriae, the uncoiling force for MrkAG189A fimbriae was not constant. The MrkAG189A fimbriae also exhibited a lower enthalpy in the differential scanning calorimetry analysis. Together, these findings indicate that the mutant fimbriae are less stable than the wild-type. This study has demonstrated that the C-terminal β strands of MrkA are required for the assembly and structural stability of fimbriae.