General metabolism of Laribacter hongkongensis: a genome-wide analysis

Background

Laribacter hongkongensis is associated with community-acquired gastroenteritis and traveler's diarrhea. In this study, we performed an in-depth annotation of the genes and pathways of the general metabolism of L. hongkongensis and correlated them with its phenotypic characteristics.

Results

The L. hongkongensis genome possesses the pentose phosphate and gluconeogenesis pathways and tricarboxylic acid and glyoxylate cycles, but incomplete Embden-Meyerhof-Parnas and Entner-Doudoroff pathways, in agreement with its asaccharolytic phenotype. It contains enzymes for biosynthesis and β-oxidation of saturated fatty acids, biosynthesis of all 20 universal amino acids and selenocysteine, the latter not observed in Neisseria gonorrhoeae, Neisseria meningitidis and Chromobacterium violaceum. The genome contains a variety of dehydrogenases, enabling it to utilize different substrates as electron donors. It encodes three terminal cytochrome oxidases for respiration using oxygen as the electron acceptor under aerobic and microaerophilic conditions and four reductases for respiration with alternative electron acceptors under anaerobic conditions. The presence of complete tetrathionate reductase operon may confer survival advantage in mammalian host in association with diarrhea. The genome contains CDSs for incorporating sulfur and nitrogen by sulfate assimilation, ammonia assimilation and nitrate reduction. The existence of both glutamate dehydrogenase and glutamine synthetase/glutamate synthase pathways suggests an importance of ammonia metabolism in the living environments that it may encounter.

Conclusions

The L. hongkongensis genome possesses a variety of genes and pathways for carbohydrate, amino acid and lipid metabolism, respiratory chain and sulfur and nitrogen metabolism. These allow the bacterium to utilize various substrates for energy production and survive in different environmental niches.     

Transport genes and chemotaxis in Laribacter hongkongensis: a genome-wide analysis

Background

Laribacter hongkongensis is a Gram-negative, sea gull-shaped rod associated with community-acquired gastroenteritis. The bacterium has been found in diverse freshwater environments including fish, frogs and drinking water reservoirs. Using the complete genome sequence data of L. hongkongensis, we performed a comprehensive analysis of putative transport-related genes and genes related to chemotaxis, motility and quorum sensing, which may help the bacterium adapt to the changing environments and combat harmful substances.

Results

A genome-wide analysis using Transport Classification Database TCDB, similarity and keyword searches revealed the presence of a large diversity of transporters (n = 457) and genes related to chemotaxis (n = 52) and flagellar biosynthesis (n = 40) in the L. hongkongensis genome. The transporters included those from all seven major transporter categories, which may allow the uptake of essential nutrients or ions, and extrusion of metabolic end products and hazardous substances. L. hongkongensis is unique among closely related members of Neisseriaceae family in possessing higher number of proteins related to transport of ammonium, urea and dicarboxylate, which may reflect the importance of nitrogen and dicarboxylate metabolism in this assacharolytic bacterium. Structural modeling of two C⁴-dicarboxylate transporters showed that they possessed similar structures to the determined structures of other DctP-TRAP transporters, with one having an unusual disulfide bond. Diverse mechanisms for iron transport, including hemin transporters for iron acquisition from host proteins, were also identified. In addition to the chemotaxis and flagella-related genes, the L. hongkongensis genome also contained two copies of qseB/qseC homologues of the AI-3 quorum sensing system.

Conclusions

The large number of diverse transporters and genes involved in chemotaxis, motility and quorum sensing suggested that the bacterium may utilize a complex system to adapt to different environments. Structural modeling will provide useful insights on the transporters in L. hongkongensis.     

Virulence determinants, drug resistance and mobile genetic elements of Laribacter hongkongensis: a genome-wide analysis

Background

Laribacter hongkongensis is associated with community-acquired gastroenteritis and traveler's diarrhea. In this study, we performed an in-depth annotation of the genes in its genome related to the various steps in the infective process, drug resistance and mobile genetic elements.

Results

For acid and bile resistance, L. hongkongensis possessed a urease gene cassette, two arc gene clusters and bile salt efflux systems. For intestinal colonization, it possessed a putative adhesin of the autotransporter family homologous to those of diffusely adherent Escherichia coli (E. coli) and enterotoxigenic E. coli. To evade from host defense, it possessed superoxide dismutase and catalases. For lipopolysaccharide biosynthesis, it possessed the same set of genes that encode enzymes for synthesizing lipid A, two Kdo units and heptose units as E. coli, but different genes for its symmetrical acylation pattern, and nine genes for polysaccharide side chains biosynthesis. It contained a number of CDSs that encode putative cell surface acting (RTX toxin and hemolysins) and intracellular cytotoxins (patatin-like proteins) and enzymes for invasion (outer membrane phospholipase A). It contained a broad variety of antibiotic resistance-related genes, including genes related to β-lactam (n = 10) and multidrug efflux (n = 54). It also contained eight prophages, 17 other phage-related CDSs and 26 CDSs for transposases.

Conclusions

The L. hongkongensis genome possessed genes for acid and bile resistance, intestinal mucosa colonization, evasion of host defense and cytotoxicity and invasion. A broad variety of antibiotic resistance or multidrug resistance genes, a high number of prophages, other phage-related CDSs and CDSs for transposases, were also identified.     

Laribacter hongkongensis: an emerging pathogen of infectious diarrhea

Laribacter hongkongensis is relatively a new name in the list of bacterial pathogens for gastroenteritis and travelers’ diarrhea. Addition of another name increases burden on the enteric infections as a whole. L. hongkongensis belongs to Neisseriaceae family of β subclass Proteobacteria. L. hongkongensis was initially isolated in Hong Kong from blood and empyema of an alcoholic cirrhotic patient in 2001, followed by reports from Korea and China, representing a total of 38 articles in PubMed until April 2013. As of now, there is no report from Indian subcontinent where infectious diarrhea is very much prevalent and a major burden. This review provides information about the microbiological characteristics, consideration of an emerging pathogen, relative pathogenicity, genome and proteome content, resistance toward multiple antibiotics, adaptability to different stress, and other features since its time of discovery. Investigation for this bacterium may avoid misidentification as other microbial flora. Further studies like the geographical distribution, type of infection, disease burden, pathogenicity, or genomic exploration of this bacterium will be useful in characterizing them properly. This bacterium may possibly be the emerging threat to public health.     

Characterization of a novel cryptic plasmid, pHLHK26, in Laribacter hongkongensis

We report the complete nucleotide sequence and characterization of a cryptic plasmid, pHLHK26, recovered from a strain of Laribacter hongkongensis isolated from a patient with community acquired gastroenteritis. pHLHK26 consists of 8700 bp, with G + C content 51.3%. The copy number (mean +/- SD) is 0.57 +/- 0.07 and it is stable after four passages (about 240 generations) in the absence of selection. There is a predicted origin of replication that consists of a DnaA box and five 22-bp direct repeats. pHLHK26 has four ORFs with two genes encoded in the sense direction and the other two in antisense direction. These four ORFs encode a putative plasmid partitioning protein of the ParA family, a putative protein that contains putative ADP-ribose 1"-phosphatase activity belonging to the Appr-1-p processing enzyme family, a putative recombinase (TniR) of the resolvase/invertase family, and a putative replication protein, respectively. We speculate that pHLHK26 is a theta, possibly Class A, replicative plasmid, as it contains an origin of replication with AT-rich region, a number of iterons and a DnaA box and a gene that encodes a replicative protein most homologous to those of other theta replicative plasmids and it shares eight of the nine positions of the consensus sequence TTAT(C/A)CA(C/A)A (TTTTCCACA in pHLHK26) in the DnaA boxes observed in other classical examples of Class A plasmids of this group.     

不同来源株香港海鸥形菌药敏试验结果分析

目的对分离自社区获得性胃肠炎患者及淡水鱼肠道内的香港海鸥形菌(Laribacter hongkongensis,LH)进行药敏试验,为临床合理使用抗菌药物提供依据。方法2005年至2006年杭州市第一人民医院肠道门诊从社区获得性胃肠炎患者中分离到4株及2种淡水鱼体内10株LH菌株,计14株,按美国CLSI推荐的纸片扩散法进行药敏试验。结果药敏结果显示,14株LH菌株对24种常用抗菌药物中的15种药物,分别表现出不同程度的耐药性,耐药率达62.5%;尤其对青霉素、头孢唑啉和头孢哌酮等3种药物,14株LH菌株均为耐药,耐药率为100.0%;其中人体株LHHZ26,对12种药物耐药、1种为中介。LH对3代头孢表现为较高的耐药性,3种头孢药物的耐药率为71.43%~100%;14株LH株对头孢吡肟及头孢哌酮/舒巴坦均为敏感。对氨曲南(14.29%)、庆大霉素(7.14%)、复方新诺明(7.14%)的耐药率较低;喹诺酮类、氯霉素、阿米卡星、美罗培南、亚胺培南等药物均敏感。结论LH菌株对β-内酰胺类药物表现出耐药性,对青霉素类、头孢菌素类药物广泛耐药。建议对LH感染患者喹诺酮类为首选治疗用药。     

Structural and functional insight into the mechanism of an alkaline exonuclease from Laribacter hongkongensis

Alkaline exonuclease and single-strand DNA (ssDNA) annealing proteins (SSAPs) are key components of DNA recombination and repair systems within many prokaryotes, bacteriophages and virus-like genetic elements. The recently sequenced β-proteobacterium Laribacter hongkongensis (strain HLHK9) encodes putative homologs of alkaline exonuclease (LHK-Exo) and SSAP (LHK-Bet) proteins on its 3.17 Mb genome. Here, we report the biophysical, biochemical and structural characterization of recombinant LHK-Exo protein. LHK-Exo digests linear double-stranded DNA molecules from their 5′-termini in a highly processive manner. Exonuclease activities are optimum at pH 8.2 and essentially require Mg²⁺ or Mn²⁺ ions. 5′-phosphorylated DNA substrates are preferred over dephosphorylated ones. The crystal structure of LHK-Exo was resolved to 1.9 Å, revealing a ‘doughnut-shaped’ toroidal trimeric arrangement with a central tapered channel, analogous to that of λ-exonuclease (Exo) from bacteriophage-λ. Active sites containing two bound Mg²⁺ ions on each of the three monomers were located in clefts exposed to this central channel. Crystal structures of LHK-Exo in complex with dAMP and ssDNA were determined to elucidate the structural basis for substrate recognition and binding. Through structure-guided mutational analysis, we discuss the roles played by various active site residues. A conserved two metal ion catalytic mechanism is proposed for this class of alkaline exonucleases.     

Construction of an inducible expression shuttle vector for Laribacter hongkongensis, a novel bacterium associated with gastroenteritis

An Escherichia coli-Laribacter hongkongensis shuttle vector (pPW380) was constructed by ligating the 4701-bp EcoRI digested fragment of pHLHK8 to EcoRI digested pBK-CMV. An E. coli-L. hongkongensis inducible expression shuttle vector was further constructed by ligating a 2105-bp fragment that contains the tetracycline repressor and tetracycline-inducible promoter region of pALC2084 to the 8897-bp fragment of pPW380, deletion of the green fluorescent protein gene, and insertion of a multiple cloning site. This inducible expression system was able to express two commonly used reporter genes, the green fluorescent protein gene and the glutathione S-transferase gene, efficiently in E. coli and L. hongkongensis.     

Isolation of Laribacter hongkongensis from Little Egrets (Egretta garzetta) in Hangzhou, China

Aims: Laribacter hongkongensis is well adapted to diverse freshwater environments and is associated with human community‐acquired gastroenteritis and traveller’s diarrhoea. The study intended to investigate whether L. hongkongensis could be detected in Little Egrets (Egretta garzetta, a widespread aquatic bird) in Hangzhou, China. Methods and Results: A total of 176 faecal specimens from three habitats in Hangzhou were obtained for this study. L. hongkongensis was isolated from 12 (6·8%) specimens. Identification of the 12 L. hongkongensis isolates was achieved through a combination of phenotypic characterization and 16S rRNA gene sequence analysis. Conclusions: The results illustrate that L. hongkongensis is present in the faeces of Little Egrets from three different habitats in Hangzhou, China. Significance and Impact of the Study: This study suggests the possibility that Little Egrets serve as mechanical carriers of L. hongkongensis, thus potentially transmitting the organism between bodies of water.     

Isolation of Laribacter hongkongensis, a novel bacterium associated with gastroenteritis, from drinking water reservoirs in Hong Kong

AIMS: Freshwater fish has been found to be the reservoir of Laribacter hongkongensis, a recently discovered bacterium associated with community-acquired gastroenteritis. However, little is known about the ecology of this bacterium in the aquatic environment. We carried out a surveillance study to investigate the presence of L. hongkongensis in water and freshwater fish from 10 drinking water reservoirs in Hong Kong. METHODS AND RESULTS: Using membrane filtration, L. hongkongensis was isolated from the waters of six reservoirs, with numbers ranging from 1 to 12 CFU l(-1). Higher recovery rates were observed in summer and during days of higher water and ambient temperatures. Of 27 freshwater fish collected from the reservoirs, L. hongkongensis was recovered from the intestines of two fish, a Goldfish and a Nile tilapia. Overall, 35 different pulsed-field gel electrophoresis patterns are found among the 59 isolates recovered from water and the two isolates from freshwater fish. CONCLUSIONS: The present report represents the first to demonstrate the presence of L. hongkongensis in natural water environments. SIGNIFICANCE AND IMPACT OF THE STUDY: Although it is unlikely that treated, drinking water is an important source of L. hongkongensis-associated gastroenteritis, one should be aware of the possibility of other contaminated water as a source of human infection.     

The Complete Genome and Proteome of Laribacter hongkongensis Reveal Potential Mechanisms for Adaptations to Different Temperatures and Habitats

Laribacter hongkongensis is a newly discovered Gram-negative bacillus of the Neisseriaceae family associated with freshwater fish–borne gastroenteritis and traveler's diarrhea. The complete genome sequence of L. hongkongensis HLHK9, recovered from an immunocompetent patient with severe gastroenteritis, consists of a 3,169-kb chromosome with G+C content of 62.35%. Genome analysis reveals different mechanisms potentially important for its adaptation to diverse habitats of human and freshwater fish intestines and freshwater environments. The gene contents support its phenotypic properties and suggest that amino acids and fatty acids can be used as carbon sources. The extensive variety of transporters, including multidrug efflux and heavy metal transporters as well as genes involved in chemotaxis, may enable L. hongkongensis to survive in different environmental niches. Genes encoding urease, bile salts efflux pump, adhesin, catalase, superoxide dismutase, and other putative virulence factors—such as hemolysins, RTX toxins, patatin-like proteins, phospholipase A1, and collagenases—are present. Proteomes of L. hongkongensis HLHK9 cultured at 37°C (human body temperature) and 20°C (freshwater habitat temperature) showed differential gene expression, including two homologous copies of argB, argB-20, and argB-37, which encode two isoenzymes of N-acetyl-L-glutamate kinase (NAGK)—NAGK-20 and NAGK-37—in the arginine biosynthesis pathway. NAGK-20 showed higher expression at 20°C, whereas NAGK-37 showed higher expression at 37°C. NAGK-20 also had a lower optimal temperature for enzymatic activities and was inhibited by arginine probably as negative-feedback control. Similar duplicated copies of argB are also observed in bacteria from hot springs such as Thermus thermophilus, Deinococcus geothermalis, Deinococcus radiodurans, and Roseiflexus castenholzii, suggesting that similar mechanisms for temperature adaptation may be employed by other bacteria. Genome and proteome analysis of L. hongkongensis revealed novel mechanisms for adaptations to survival at different temperatures and habitats.     

Environmental robustness and the adaptability of populations

Recent work has shown that genetic robustness can either facilitate or impede adaptation. But the impact of environmental robustness on adaptation remains unclear. Environmental robustness helps ensure that organisms consistently develop the same phenotype in the face of "environmental noise" during development. Under purifying selection, those genotypes that express the optimal phenotype most reliably will be selectively favored. The resulting reduction in genetic variation tends to slow adaptation when the population is faced with a novel target phenotype. However, environmental noise sometimes induces the expression of an alternative advantageous phenotype, which may speed adaptation by genetic assimilation. Here, we use a population-genetic model to explore how these two opposing effects of environmental noise influence the capacity of a population to adapt. We analyze how the rate of adaptation depends on the frequency of environmental noise, the degree of environmental robustness in the population, the distribution of environmental robustness across genotypes, the population size, and the strength of selection for a newly adaptive phenotype. Over a broad regime, we find that environmental noise can either facilitate or impede adaptation. Our analysis uncovers several surprising insights about the relationship between environmental noise and adaptation, and it provides a general framework for interpreting empirical studies of both genetic and environmental robustness.     

GCTA: a tool for genome-wide complex trait analysis

For most human complex diseases and traits, SNPs identified by genome-wide association studies (GWAS) explain only a small fraction of the heritability. Here we report a user-friendly software tool called genome-wide complex trait analysis (GCTA), which was developed based on a method we recently developed to address the "missing heritability" problem. GCTA estimates the variance explained by all the SNPs on a chromosome or on the whole genome for a complex trait rather than testing the association of any particular SNP to the trait. We introduce GCTA's five main functions: data management, estimation of the genetic relationships from SNPs, mixed linear model analysis of variance explained by the SNPs, estimation of the linkage disequilibrium structure, and GWAS simulation. We focus on the function of estimating the variance explained by all the SNPs on the X chromosome and testing the hypotheses of dosage compensation. The GCTA software is a versatile tool to estimate and partition complex trait variation with large GWAS data sets.     

Identification and classification of genes required for tolerance to high-sucrose stress revealed by genome-wide screening of Saccharomyces cerevisiae

Yeasts used in bread making are exposed to high concentrations of sucrose during sweet dough fermentation. Despite its importance, tolerance to high-sucrose stress is poorly understood at the gene level. To clarify the genes required for tolerance to high-sucrose stress, genome-wide screening was undertaken using the complete deletion strain collection of diploid Saccharomyces cerevisiae. The screening identified 273 deletions that yielded high sucrose sensitivity, approximately 20 of which were previously uncharacterized. These 273 deleted genes were classified based on their cellular function and localization of their gene products. Cross-sensitivity of the high-sucrose-sensitive mutants to high concentrations of NaCl and sorbitol was studied. Among the 273 sucrose-sensitive deletion mutants, 269 showed cross-sensitivities to sorbitol or NaCl, and four (i.e. ade5,7, ade6, ade8, and pde2) were specifically sensitive to high sucrose. The general stress response pathways via high-osmolarity glycerol and stress response element pathways and the function of the invertase in the ade mutants were similar to those in the wild-type strain. In the presence of high-sucrose stress, intracellular contents of ATP in ade mutants were at least twofold lower than that of the wild-type cells, suggesting that depletion of ATP is a factor in sensitivity to high-sucrose stress. The genes identified in this study might be important for tolerance to high-sucrose stress, and therefore should be target genes in future research into molecular modification for breeding of yeast tolerant to high-sucrose stress.     

Quantitative genome-wide analysis of yeast deletion strain sensitivities to oxidative and chemical stress

Understanding the actions of drugs and toxins in a cell is of critical importance to medicine, yet many of the molecular events involved in chemical resistance are relatively uncharacterized. In order to identify the cellular processes and pathways targeted by chemicals, we took advantage of the haploid Saccharomyces cerevisiae deletion strains (Winzeler et al., 1999). Although ~4800 of the strains are viable, the loss of a gene in a pathway affected by a drug can lead to a synthetic lethal effect in which the combination of a deletion and a normally sublethal dose of a chemical results in loss of viability. WE carried out genome-wide screens to determine quantitative sensitivities of the deletion set to four chemicals: hydrogen peroxide, menadione, ibuprofen and mefloquine. Hydrogen peroxide and menadione induce oxidative stress in the cell, whereas ibuprofen and mefloquine are toxic to yeast by unknown mechanisms. Here we report the sensitivities of 659 deletion strains that are sensitive to one or more of these four compounds, including 163 multichemicalsensitive strains, 394 strains specific to hydrogen peroxide and/or menadione, 47 specific to ibuprofen and 55 specific to mefloquine.We correlate these results with data from other large-scale studies to yield novel insights into cellular function.