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 of the tyrosine phenol lyase gene from Citrobacter freundii

Summary The gene for the enzyme tyrosine phenol-lyase (TPL) was initially isolated on a 45 kbp fragment of Citrobacter freundii genomic DNA contained in a cosmid. Subsequent restriction enzyme digestion and sub-cloning resulted in the gene being contained on a 2.4 kbp DNA fragment.     

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-Asparagainases from Citrobacter freundii.

Three enzymes which catalyze the hydrolysis of L-asparagine have been identified in extracts of Citrobacter freundii. One of these (asparaginase-glutaminase (EC 3.5.1.1) also shows substantial glutaminase activity. This enzyme is extremely labile, is sensitive to inactivation by p-chloromercuribenzoate, and is not protected by dithiothreitol. A second enzyme (asparaginase B) is also sensitive to mercurials but is protected from inactivation by dithiothreitol. This enzyme has a relatively low affinity for L-asparagine (Km = 1.7-10(-3) M). The third enzyme (asparaginase A) is insensitive to inactivation by mercurials, is stable upon long term storage and has a relatively high affinity for L-asparagine (Km = 2.9-10(-5) M). This enzyme has been purified to homogeneity and has a molecular weight of approx. 140 000; the subunit weight being approx. 33 000. The C. freundii asparaginase A produced significant increases in the survival time of C3H/HE mice carrying the 6C3HED lymphoma tumor.     

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.     

Recovery of heat-injured Citrobacter freundii cells

The influence of various factors in the recovery process of heat-injured cells of Citrobacter freundii has been studied. In particular the temperature of the liquid recovery medium and the residence time of the cells in this medium are important. Cells heated in media with a high osmotic pressure are better recovered in low osmotic liquid recovery media. The influence of the temperature of the solid recovery medium on the decimal reduction time for Ctbt. freundii cells strongly suggests that the number of critical sites to be inactivated before the cell wall be lethally injured also depends on the recovery conditions.     

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.     

Growth conditions and heat resistance of Citrobacter freundii

The influence of the growth medium and the growth temperature on the heat resistance of Citrobacter freundii has been established. Logarithmic growth phase cells grown on rich media have a higher heat resistance than cells of the same phase grown on minimal media. This finding was independent of type of carbon source in the growth medium, but the kind of carbon source has a definite influence on the heat resistance. Logarithmic phase cells grown at 37°C are much more heat stable than cells grown at 20 or 41°C. Stationary growth phase cells are much more heat resistant than logarithmic phase cells, whereas Mg²⁺-or glucose-starved cells are even slightly more heat stable than stationary phase cells.     

Properties of free and bound Citrobacter freundii lipopolysaccharides

Culture medium content of free lipopolysaccharide (LPS) components spontaneously released from a Citrobacter freundii culture grown in minimum synthetic medium was determined during early (8-hr culture) and late (24-hr culture) phases of growth. As judged by Limulus-lysate test, free LPS occurred in the medium as early as after 8 hrs of incubation, i.e. at the beginning of log growth phase. As the culture continued to grow the LPS amount released into culture medium kept rising, reaching 30% of endotoxin present in 24-hr Citrobacter culture. The released LPS complex was isolated by separation and its physicochemical, immunochemical and biological properties were determined and compared with those of cell-bound endotoxin recovered from cells by phenol extraction. Comparisons revealed distinct differences in the chemical composition and the degree of heterogeneity; free LPS was less heterogeneous. Immunologically, free LPS differed from bound LPS in the structure of macromolecules, but was identical with it in some antigenic determinants. The biological activity of free LPS preparation was greater than that of cell-bound LPS.     

Characterization of swarming motility in Citrobacter freundii

Bacterial swarming motility is a flagella-dependent translocation on the surface environment. It has received extensive attention as a population behavior involving numerous genes. Here, we report that Citrobacter freundii, an opportunistic pathogen, exhibits swarming movement on a solid medium surface with appropriate agar concentration. The swarming behavior of C. freundii was described in detail. Insertional mutagenesis with transposon Mini-Tn5 was carried out to discover genetic determinants related to the swarming of C. freundii. A number of swarming genes were identified, among which flhD, motA, motB, wzx, rfaL, rfaJ, rfbX, rfaG, rcsD, rcsC, gshB, fabF, dam, pgi, and rssB have been characterized previously in other species. In mutants related to lipopolysaccharide synthesis and RcsCDB signal system, a propensity to form poorly motile bacterial aggregates on the agar surface was observed. The aggregates hampered bacterial surface migration. In several mutants, the insertion sites were identified to be in the ORF of yqhC, yeeZ, CKO_03941, glgC, and ttrA, which have never been shown to be involved in swarming. Our results revealed several novel characteristics of swarming motility in C. freundii which are worthy of further study.     

A new site-specific endodeoxyribonuclease from Citrobacter freundii

Cfr10 I, a site-specific endonuclease from Citrobacter freundii strain RFL10, was isolated. It recognizes and cleaves the family of related sequences: 5'Pu decreases CCGGPy to generate DNA fragments with 5' tetranucleotide extensions. Cfr10 I may be useful in molecular cloning experiments, especially in conjunction with other enzymes which generate the same terminal extensions.     

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.     

A specific cephalosporin-binding protein of Citrobacter freundii.

1. A cephalosporin-binding protein obtained from a strain of Citrobacter freundii was purified to the extent of a single band in analytical and sodium dodecyl sulfate-containing disc electrophoresis. 2. The molecular weight determined by disc electrophoresis was 53 000. 3. The binding protein did not show any beta-lactamase activity at substrate concentrations examined: 6 mM to 100 muM of penicillins and 12 mM to 100 muM of cephalosporins. 4. In gel filtration, [14C]benzylpenicillin was found not to bind to the binding protein. 5. In fluorescence titration, all cephalosporins tested quenched the fluorescence. Association constants of cephalosporins were in the range of 0.8-12-103 M-1, and one binding site was calculated for all cephalosporins tested.     

Mutants of Citrobacter freundii That Transport and Utilize Melibiose

We have isolated mutants of Citrobacter freundii that can grow on melibiose. Inducible α-galactosidase activity and melibiose transport activity were detected in the mutant cells but not in the wild-type cells. We detected a DNA region which hybridized with melB (the gene for the melibiose transporter) DNA of Escherichia coli in the chromosomal DNA of wild-type C. freundii. Protons, but not sodium ions, were found to be the coupling cations for melibiose (and methyl-β-d-thiogalactoside) transport in the mutant cells.

The melibiose transporter of Escherichia coli is a secondary transporter which mediates symport of monovalent cations and melibiose or its analogs (16). This transporter is a valuable system for the investigation of structure-function relationships in a cation-coupled symporter. Either Na⁺, H⁺, or Li⁺ is utilized as a coupling cation for transport of melibiose or other galactosides (or galactose). The coupling cation utilized varies depending on the substrate transported (16). Na⁺ is the most effective coupling cation for melibiose transport, followed by H⁺ and Li⁺ (Li⁺ is a poor coupling cation). With methyl-β-d-thiogalactoside (TMG) as the substrate, both Na⁺ and Li⁺, but not H⁺, are utilized (5, 16). We cloned the gene (melB) encoding the melibiose transporter and sequenced it (3, 18). Thus, the primary structure of the melibiose transporter (MelB) was deduced. Mutational analysis revealed many amino acid residues that are important for the function of the melibiose transporter, especially for cation recognition (11, 17).Analyses of functionally and structurally related proteins are valuable for the understanding of structure-function relationships in the proteins. Several microorganisms possess melibiose transporters. The melibiose transporters from Salmonella typhimurium (6), Klebsiella pneumoniae (2), Enterobacter aerogenes (9), and Enterobacter cloacae (8), in addition to E. coli, have been characterized and sequenced (13). Such analyses are also useful for understanding the evolutionary relationships of the transporters (and microorganisms).Citrobacter freundii is a member of the Enterobacteriaceae and is often found in clinical specimens as an opportunistic or secondary pathogen (12). Although cells of C. freundii are able to utilize lactose as a carbon source (10), they are unable to utilize melibiose. Here we report the isolation of C. freundii mutants able to grow on melibiose. We also describe the properties of the melibiose transporter in the mutants.Isolation of mutants.Cells of C. freundii ATCC 8090 grown in L medium (4) were densely streaked on agar plates containing a minimal medium (14) supplemented with 10 mM melibiose. Na⁺ salts in the minimal medium were replaced with K⁺ salts. After incubation at 37°C for 2 days, colonies appeared on the plates. Since these mutant cells utilized melibiose as a carbon source, they must have expressed a transporter for melibiose and an enzyme for the degradation of melibiose. We isolated the colonies and purified them on agar plates containing minimal medium and melibiose. Thereafter, we measured the growth of two of the mutants, M4 and M7, on melibiose. The mutant cells grew well on melibiose, although the wild-type cells did not (data not shown). Cells of M4 showed better growth than cells of M7. The generation time for M7 was about 1.5 times longer than that for M4.α-Galactosidase activity in the mutants.Wild-type and mutant cells of C. freundii were grown in minimal medium supplemented with 1% tryptone either in the absence or presence of 10 mM melibiose at 37°C under aerobic conditions, and α-galactosidase activity was measured as described previously (15). As shown in Table ​Table1,1, cells of the wild type and M7 grown in the absence of melibiose had no α-galactosidase activity. Cells of M4 grown in the absence of melibiose, however, showed some α-galactosidase activity. When grown in the presence of melibiose, cells of M4 showed very high α-galactosidase activity, cells of M7 showed moderate activity, and wild-type cells showed no activity. Thus, cells of M4 and M7 possessed inducible α-galactosidase activities, although the activity was partially constitutive in M4 cells (Table ​(Table1).1). TABLE 1α-Galactosidase activity in wild-type and mutant cells of C. freundii

Characterization of Membrane-bound Spermidine Dehydrogenase of Citrobacter freundii

Spermidine dehydrogenase found in the membrane fraction of Citrohacter freundii IFO 12681 was solubilized with Triton X-100 and further purified to homogeneity. The properties of the membrane enzyme were almost identical to those obtained from the soluble fraction of the organism with respect to molecular and catalytic properties. Thus, binding properties of the enzyme to the bacterial membrane were checked. The ratio of enzyme activity found in the soluble fraction to the membrane fraction was dependent on salt concentration during cell disruption. A hydrophobic interaction was largely involved in anchoring the enzyme to the membrane fraction. Purified spermidine dehydrogenase from the soluble fraction was readily adsorbed into the membrane fraction in the presence of salt. Spermidine dehydrogenase appeared to be a membrane-bound enzyme localized in the cytoplasmic membranes in a manner that makes a partial release of the enzyme possible during mechanical cell disruption. When spermidine oxidation was done with the resting cells of C. freundii, a stoichiometric formation of two reaction products, 1,3-diaminopropane and γ-aminobutyraldeyde, was observed without any lag time. These facts indicate that the enzyme is localized on the outer surface of the cytoplasmic membranes or in the periplasmic space of the organism.     

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）。     

Synthesis of tyrosine and 3,4-dihydroxyphenylalanine by bacteria Citrobacter freundii

Among facultative-anaerobic bacteria utilizing formic acid, a large number of strains having tyrosine phenol lyase were found. The enzyme can catalyze synthesis of tyrosine and 3,4-dihydroxy phenyl alanine (DOPA) from pyruvate, ammonium and, accordingly, phenol and pyrocatechol. These strains were identified as Citrobacter freundii. Cell suspensions of the most active strains synthesized up to 75 g/l tyrosine for 12 hr, up to 86 g/l tyrosine for 24 hr, and up to 29 g/l DOPA for 42 hr. A medium containing yeast autolysate grown on hydrocarbons can be recommended to produce cells having a high tyrosine phenol lyase activity.