一株弗劳地枸橼酸杆菌中检出新ampC基因

目的探讨1株耐亚胺培南弗劳地枸橼酸杆菌的耐药机制。方法采用浓度梯度法（Etest）检测对抗菌药物的最低抑菌浓度（MIC）。通过金属酶初筛试验（协同法）检测金属酶;改良Hoged试验检测碳青霉烯酶;头孢西丁三维试验检测AmpC酶;聚合酶链反应（PCR）检测耐药基因;DNA测序决定基因型;接合试验检测耐药基因的转移性。结果弗劳地枸橼酸杆菌临床分离株NC118对亚胺培南的MIC为〉16μg/ml,金属酶初筛试验阴性,Hoged表型确证试验碳青霉烯酶阳性,AmpC酶阳性,PCR扩增及测序显示含有blaKPC-2、blaAmpC基因,该菌株所产AmpC酶基因与CMY-45型AmpC酶（GenBank：ACU00152.1）比较有5个氨基酸发生了改变,该blaCMY-2-like基因为一个新型的AmpC酶基因。结论在弗劳地枸橼酸杆菌中发现一种新的ampC基因（blaCMY-49）。     

A multigene family encoding several "finger" structures is present and differentially active in mammalian genomes

Mouse genomic DNA contains multiple copies of sequences homologous to the Drosophila "Krüppel," a member of the "gap" class of developmental control genes of the fruit fly. The most interesting aspect of the homologous region is that, like Xenopus TFIIIA, it contains multiple finger-like folded domains capable of binding to nucleic acids. We have isolated six individual phages from a mouse genomic library on the basis of their DNA homology to Krüppel finger-coding probes, and describe here the DNA sequence and expression of two such clones containing finger-like structures. Upon differentiation of mouse teratocarcinoma cell line F9 with retinoic acid and cAMP, the expression of both genes was drastically reduced, and in one instance was undetectable. Each of the several other eukaryotic DNAs analyzed contained multiple copies of homologous genes with putative finger structures, indicating the presence of a finger-containing multigene family in higher organisms.     

Isolation and characterization of Hfr males in Citrobacter freundii

Citrobacter freundii Hfr donor strains were isolated from a C. freundii strain harbouring a temperature-sensitive factor F ts 114 lac ⁺, by selecting for integrative suppression of the ts 114 mutation. Three Hfr strains were characterized, which transfer their chromosomes in a linear and oriented order. The first strain transfers: O-aro ⁺-ilv ⁺-pur ⁺-thr ⁺-leu ⁺-pro ⁺, the second: O-ilv ⁺-pur ⁺-thr ⁺-leu ⁺-pro ⁺ and the third: O-ilv ⁺-aro ⁺-nad ⁺-his ⁺-pro ⁺. The whole chromosome is transferred into the recipient cell within about 145 minutes. From these results we concluded that the linkage map of C. freundii is circular. Mating-pair formation on a membrane filter resulted in more recombinants being formed as compared with mating-pair formation in liquid medium. Furthermore the mating-pairs formed on a membrane were more stable. From one Hfr strain heterogenic F-prime factors could be isolated bearing the F ts 114 lac ⁺ genes from Escherichia coli and the pur ⁺ and/or ilv ⁺ genes from C. freundii. Preliminary mapping by interrupted mating indicated that the linkage map of C. freundii is in general very similar to those of E. coli, Salmonella typhimurium and Klebsiella aerogenes.     

Isolation and characterization of cytotoxic, aggregative Citrobacter freundii

Citrobacter freundii is an infrequent but established cause of diarrhea in humans. However, little is known of its genetic diversity and potential for virulence. We analyzed 26 isolates, including 12 from human diarrheal patients, 2 from human fecal samples of unknown diarrheal status, and 12 from animals, insects, and other sources. Pulsed field gel electrophoresis using XbaI allowed us to divide the 26 isolates into 20 pulse types, while multi-locus sequence typing using 7 housekeeping genes allowed us to divide the 26 isolates into 6 sequence types (STs) with the majority belonging to 4 STs. We analyzed adhesion and cytotoxicity to HEp-2 cells in these 26 strains. All were found to adhere to HEp-2 cells. One strain, CF74, which had been isolated from a goat, showed the strongest aggregative adhesion pattern. Lactate dehydrogenase (LDH) released from HEp-2 cells was evaluated as a measure of cytotoxicity, averaging 7.46%. Strain CF74 induced the highest level of LDH, 24.3%, and caused >50% cell rounding, detachment, and death. We named strain CF74 "cytotoxic and aggregative C. freundii." Genome sequencing of CF74 revealed that it had acquired 7 genomic islands, including 2 fimbriae islands and a type VI secretion system island, all of which are potential virulence factors. Our results show that aggregative adherence and cytotoxicity play an important role in the pathogenesis of C. freundii.     

Purification and characterization of selenocysteine beta-lyase from Citrobacter freundii.

The purification and characterization of bacterial selenocysteine beta-lyase, an enzyme which specifically catalyzes the cleavage of L-selenocysteine to L-alanine and Se0, are presented. The enzyme, purified to near homogeneity from Citrobacter freundii, is monomeric with a molecular weight of ca. 64,000 and contains 1 mol of pyridoxal 5'-phosphate as a cofactor per mol of enzyme. L-Selenocysteine is the sole substrate (Km, 0.95 mM). L-Cysteine is a competitive inhibitor of the enzyme (Ki, 0.65 mM). The enzyme also catalyzes the alpha, beta elimination of beta-chloro-L-alanine to form NH3, pyruvate, and Cl- and is irreversibly inactivated during the reaction. The physicochemical properties, e.g., amino acid composition and subunit structure, of the bacterial enzyme are fairly different from those of the pig liver enzyme (Esaki et al., J. Biol. Chem. 257:4386-4391, 1982). However, the catalytic properties of both enzymes, e.g., substrate specificity and inactivation by the substrate or a mechanism-based inactivator, beta-chloro-L-alanine, are very similar.     

Expression, purification and characterization of flavin reductase from Citrobacter freundii A1

Flavin reductase plays an important biological role in catalyzing the reduction of flavin by NAD(P)H oxidation. The gene that codes for flavin reductase from Citrobacter freundii A1 was cloned and expressed in Escherichia coli BL21(DE3)pLysS. In this study, we aimed to characterize the purified recombinant flavin reductase of C. freundii A1. The recombinant enzyme was purified to homogeneity and the biochemical profiles, including the effect of pH, temperature, metal ions and anions on flavin reductase activity and stability, were determined. This enzyme exhibited optimum activity at 45 °C in a 10-min reaction at pH 7.5 and was stable at temperatures up to 30 °C. At 0.1 mM concentration of metal ions, flavin reductase activity was stimulated by divalent cations including Mn²⁺, Sr²⁺, Ni²⁺, Sn²⁺, Ba²⁺, Co²⁺, Mg²⁺, Ca²⁺ and Pb²⁺. Ag⁺ was noticeably the strongest inhibitor of recombinant flavin reductase of C. freundii A1. This enzyme should not be defined as a standard flavoprotein. This is the first attempt to characterize flavin reductase of C. freundii origin.     

L-methionine degradation pathway in Kluyveromyces lactis: identification and functional analysis of the genes encoding L-methionine aminotransferase

Kluyveromyces lactis is one of the cheese-ripening yeasts and is believed to contribute to the formation of volatile sulfur compounds (VSCs) through degradation of L-methionine. L-methionine aminotransferase is potentially involved in the pathway that results in the production of methanethiol, a common precursor of VSCs. Even though this pathway has been studied previously, the genes involved have never been studied. In this study, on the basis of sequence homology, all the putative aminotransferase-encoding genes from K. lactis were cloned in an overproducing vector, pCXJ10, and their effects on the production of VSCs were analyzed. Two genes, KlARO8.1 and KlARO8.2, were found to be responsible for L-methionine aminotransferase activity. Transformants carrying these genes cloned in the pCXJ10 vector produced threefold-larger amounts of VSCs than the transformant containing the plasmid without any insert or other related putative aminotransferases produced.     

Pathway redesign for deoxyviolacein biosynthesis in Citrobacter freundii and characterization of this pigment

Violacein (Vio) is an important purple pigment with many potential bioactivities. Deoxyviolacein, a structural analog of Vio, is always synthesized in low concentrations with Vio in wild-type bacteria. Due to deoxyviolacein's low production and difficulties in isolation and purification, little has been learned regarding its function and potential applications. This study was the first effort in developing a stable and efficient biosynthetic system for producing pure deoxyviolacein. A recombinant plasmid with vioabce genes was constructed by splicing using an overlapping extension-polymerase chain reaction, based on the Vio-synthesizing gene cluster of vioabcde, originating from Duganella sp. B2, and was introduced into Citrobacter freundii. With the viod gene disrupted in the Vio synthetic pathway, Vio production was completely abolished and the recombinant C. freundii synthesized only deoxyviolacein. Interestingly, vioe gene expression was strongly stimulated in the viod-deleted recombinant strain, indicating that viod disruptions could potentially induce polar effects upon the downstream vioe gene within this small operon. Deoxyviolacein production by this strain reached 1.9 g/L in shaker flasks. The product exhibited significant acid/alkali and UV resistance as well as significant inhibition of hepatocellular carcinoma cell proliferation at low concentrations of 0.1-1 μM. These physical characteristics and antitumor activities of deoxyviolacein contribute to illuminating its potential applications.     

The Yersinia high-pathogenicity island is present in different members of the family Enterobacteriaceae

A pathogenicity island termed high-pathogenicity island (HPI) is present in pathogenic Yersinia. This 35 to 45 kb island carries genes involved in synthesis, regulation and transport of the siderophore yersiniabactin. Recently, the HPI was also detected in various strains of Escherichia coli. In this study, the distribution of the HPI in the family Enterobacteriaceae was investigated. Among the 67 isolates pertaining to 18 genera and 52 species tested, nine (13.4%) harbored the island. These isolates were three E. coli, one Citrobacter diversus and five Klebsiella of various species (Klebsiella pneumoniae, Klebsiella rhinoscleromatis, Klebsiella ozaenae, Klebsiella planticola, and Klebsiella oxytoca). As in Yersinia sp., all nine isolates synthesized the HPI-encoded iron-repressible proteins HMWP1 and HMWP2. In the K. oxytoca strain, the right-end portion of the HPI was deleted, whereas the entire core region of the island was present in the eight other enterobacteria strains analyzed. In most of these isolates, the HPI was bordered by an asn tRNA locus, as in Yersinia sp. This report thus demonstrates the spread of the HPI among various members of the family Enterobacteriaceae.     

患病大鲵中弗氏柠檬酸杆菌的分离与鉴定

【目的】确定导致大鲵(Andrias davidianus)细菌性感染死亡的病原。【方法】从大鲵肝脏中分离细菌,通过Biolog微生物自动鉴定系统及分子生物学方法对纯培养的细菌进行鉴定,再用大鲵和鲫鱼分别进行人工感染试验,以确定分离菌的致病性,同时对分离到的病原菌进行药物敏感试验。【结果】从患病大鲵肝脏中分离到一株致病菌JZ01,经人工感染健康大鲵,可复制与自然发病相同的症状,且从人工感染病鲵体内再次分离到相同的病原菌。该致病菌对健康鲫鱼也有致病性。经Biolog微生物自动鉴定系统的鉴定,以及进一步的16S rDNA基因序列和系统发育分析都表明,此致病菌为弗氏柠檬酸杆菌。药物敏感性试验表明,该菌株对氨曲南、头孢三嗪、先锋噻肟等9种药物高度敏感。【结论】弗氏柠檬酸杆菌是大鲵的一种致病菌。本文在国内外首次报道了该菌对大鲵具有致病性。     

Isolation and identification of a bacteriocin with antibacterial and antibiofilm activity from Citrobacter freundii

Multi- and pan-antibiotic-resistant bacteria area major health challenge in hospital settings. Furthermore,when susceptible bacteria establish surface-attached biofilm populations, they become recalcitrant to antimicrobial therapy. Therefore, there is a need for novel antimicrobials that are effective against multi-drug-resistant and surface-attached bacteria. A screen to identify prokaryote-derived antimicrobials from a panel of over 100 bacterial strains was performed. One compound isolated from Citrobacter freundii exhibited antimicrobial activity against a wide range of Gram-negative bacteria and was effective against biofilms. Random transposon mutagenesis was performed to find mutants unable to produce the antimicrobial compound.Transposons mapped to a bacteriocin gene located on a small plasmid capable of replication in Escherichia coli. The plasmid was sequenced and found to be highly similar to a previously described colicinogenic plasmid.Expression of the predicted bacteriocin immunity gene conferred bacteriocin immunity to E. coli. The predicted bacteriocin gene, colA-43864, expressed in E. coli was sufficient to generate anti-microbial activity, and purified recombinant ColA-43864 was highly effective in killing E. coli, Citrobacter species, and Klebsiella pneumoniae cells in a planktonic and biofilm state. This study suggests that bacteriocins can be an effective way to control surface-attached pathogenic bacteria.     

Biosynthesis and export of colicin A in Citrobacter freundii CA31

Synthesis of colicin A after induction with mitomycin C was studied. Specific inhibition of chromosomal protein synthesis occurred very shortly after mitomycin addition. There was no coordinate synthesis of colicin A (61000 Mr) and low-molecular-weight protein. Free and membrane-bound polysome fractions were isolated from cells induced with mitomycin C. Colicin A is synthesized in vitro in the free polysomes and not in the membrane-bound polysomes. Conditions are described which allow a practically specific labelling of colicin A in vivo. By using this system it was possible to demonstrate that colicin A is not transferred cotranslationally across the cytoplasmic membrane. In contrast, this protein leaves the cell where it was made long after synthesis. Preliminary evidence, suggesting that pauses occur during synthesis of colicin A, is presented.     

Maize protein phosphatase gene family: identification and molecular characterization

Background

Protein phosphatases (PPs) play critical roles in various cellular processes through the reversible protein phosphorylation that dictates many signal transduction pathways among organisms. Recently, PPs in Arabidopsis and rice have been identified, while the whole complement of PPs in maize is yet to be reported.

Results

In this study, we have identified 159 PP-encoding genes in the maize genome. Phylogenetic analyses categorized the ZmPP gene family into 3 classes (PP2C, PTP, and PP2A) with considerable conservation among classes. Similar intron/exon structural patterns were observed in the same classes. Moreover, detailed gene structures and duplicative events were then researched. The expression profiles of ZmPPs under different developmental stages and abiotic stresses (including salt, drought, and cold) were analyzed using microarray and RNA-seq data. A total of 152 members were detected in 18 different tissues representing distinct stages of maize plant developments. Under salt stress, one gene was significantly up-expressed in seed root (SR) and one gene was down-expressed in primary root (PR) and crown root (CR), respectively. As for drought stress condition, 13 genes were found to be differentially expressed in leaf, out of which 10 were up-regulated and 3 exhibited down-regulation. Additionally, 13 up-regulated and 3 down-regulated genes were found in cold-tolerant line ETH-DH7. Furthermore, real-time PCR was used to confirm the expression patterns of ZmPPs.

Conclusions

Our results provide new insights into the phylogenetic relationships and characteristic functions of maize PPs and will be useful in studies aimed at revealing the global regulatory network in maize abiotic stress responses, thereby contributing to the maize molecular breeding with enhanced quality traits.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-773) contains supplementary material, which is available to authorized users.     

Protozoan genomes: gene identification and annotation

The draft sequence of several complete protozoan genomes is now available and genome projects are ongoing for a number of other species. Different strategies are being implemented to identify and annotate protein coding and RNA genes in these genomes, as well as study their genomic architecture. Since the genomes vary greatly in size, GC-content, nucleotide composition, and degree of repetitiveness, genome structure is often a factor in choosing the methodology utilised for annotation. In addition, the approach taken is dictated, to a greater or lesser extent, by the particular reasons for carrying out genome-wide analyses and the level of funding available for projects. Nevertheless, these projects have provided a plethora of material that will aid in understanding the biology and evolution of these parasites, as well as identifying new targets that can be used to design urgently required drug treatments for the diseases they cause.     

A gene encoding an SOS inhibitor is present in different conjugative plasmids.

In 9 of 20 conjugative plasmids of different incompatibility groups, including F and R100 (or R6-5), coexist two sequences which are homologous, respectively, to the gene psiB, which encodes an inhibitor of SOS induction, and to the gene ssb, which encodes a single-stranded-DNA-binding protein.     

Characterization of the Citrobacter freundii phoE gene and development of C. freundii-specific oligonucleotides

The phoE gene of Citrobacter freundii, encoding a pore-forming outer membrane protein, was cloned and its nucleotide sequence was determined. The homologies in terms of identical amino acids between the C. freundii PhoE protein and those of Escherichia coli, E. cloacae and Klebsiella pneumoniae were 90%, 86% and 84%, respectively. Two synthetic oligonucleotides, corresponding to hypervariable, cell surface-exposed regions of the protein, were tested for their specificity in polymerase chain reactions. They were specific for the species C. freundii, i.e., no reaction was detected with 35 non-C. freundii strains tested, including 17 Salmonella, two C. amalonaticus and three C. diversus strains, whereas all five C. freundii strains tested were correctly recognized.     

Biochemical and molecular characterization of the oxidative branch of glycerol utilization by Citrobacter freundii.

Glycerol dehydrogenase (EC 1.1.1.6) and dihydroxyacetone kinase (EC 2.7.1.29) were purified from Citrobacter freundii. The dehydrogenase is a hexamer of a polypeptide of 43,000 Da. The enzyme exhibited a rather broad substrate specificity, but glycerol was the preferred substrate in the physiological direction. The apparent Kms of the enzyme for glycerol and NAD+ were 1.27 mM and 57 microM, respectively. The kinase is a dimer of a polypeptide of 57,000 Da. The enzyme was highly specific for the substrates dihydroxyacetone and ATP; the apparent Kms were 30 and 70 microM, respectively. The DNA region which contained the genes encoding glycerol dehydrogenase (dhaD) and dihydroxyacetone kinase (dhaK) was cloned and sequenced. Both genes were identified by N-terminal sequence comparison. The deduced dhaD gene product (365 amino acids) exhibited high degrees of homology to glycerol dehydrogenases from other organisms and less homology to type III alcohol dehydrogenases, whereas the dhaK gene product (552 amino acids) revealed no significant homology to any other protein in the databases. A large gene (dhaR) of 1,929 bp was found downstream from dhaD. The deduced gene product (641 amino acids) showed significant similarities to members of the sigma 54 bacterial enhancer-binding protein family.     

Cloning and expression of the gene(s) for cephalosporinase production of Citrobacter freundii

The chromosomal gene(s) for cephalosporinase production of Citrobacter freundii GN346 has been cloned into vector plasmid pMK1, initially as a 7.3 kb EcoRI fragment. From the substrate profile and the response to anti-GN346 CSase serum of the enzyme produced, it was confirmed that the hybrid plasmid (pTY71) carries the relevant chromosomal cephalosporinase gene from C. freundii GN346. A restriction endonuclease cleavage map of cloned EcoRI fragments was constructed, and the structural gene of the cephalosporinase could be limited in the 1.5 kb BamHI fragment. The cloned gene(s) was expressed at an extremely low level in Escherichia coli. Furthermore, its expression was constitutive in E. coli, although inducible in its own cytoplasm.