Distribution of chitin/chitosan-like bioflocculant-producing potential in the genus Citrobacter

Some strains belonging to the genera Citrobacter and Enterobacter have been reported to produce chitin/chitosan-like bioflocculants (BFs) from acetate. In this study, to investigate the distribution of the BF-producing potential in the genus Citrobacter and to screen stably and highly BF-producing strains, we obtained 36 Citrobacter strains from different culture collection centers, which were distributed among seven species in the genus, and tested for the flocculating activities of their culture supernatants using a kaolin suspension method. As a result, 21 strains belonging to C. freundii (17 strains in 23 strains tested), C. braakii (two in two), C. youngae (one in one), and C. werkmanii (one in two) showed flocculating activity, but this ability was limited to cells grown on acetate. Gas chromatography/mass spectrometry (GC/MS) analysis of the hydrolysates from the BFs of five selected strains indicated that they consisted of glucosamine and/or N-acetylglucosamine, such as the chitin/chitosan-like BF (BF04) produced by Citrobacter sp. TKF04 (Fujita et al. J Biosci Bioeng 89: 40–46, 2000). Gel filtration chromatography using a high-performance liquid chromatography system revealed that the molecular weight ranges of these BFs varied, but the average sizes were all above 1.66?×?10⁶?Da.     

Detection and Quantification of Citrobacter freundii and C. braakii by 5′-Nuclease Polymerase Chain Reaction

A new 5′-nuclease polymerase chain reaction (PCR) system for the detection and quantification of Citrobacter freundii and C. braakii was developed with primers and the probe oriented to a specific region of the cfa gene encoding a cyclopropane fatty acid synthase. The qualitative variant of the method consisted of a conventional PCR with end-point fluorimetry or agarose gel electrophoresis, and the quantitative variant used kinetic real-time PCR measurement. The PCR system was specific for C. freundii and C. braakii, detecting neither other Citrobacter spp. nor other enteric bacteria (Escherichia coli, Salmonella enterica, and others). The detection limit of the qualitative variant of the method was 10³ cfu/mL when the amplification was followed by fluorimetry and 10⁴ cfu/mL when the amplification was followed by gel electrophoresis. The real-time PCR variant of the method facilitated quantification over a range of concentrations from 10² to 10⁸ cfu/mL, with Escherichia coli (10⁶ cfu/mL) and Salmonella enterica (10⁶ cfu/mL) having no effect on the quantification.     

Fermentation of glycerol to 1,3-propanediol by Klebsiella and Citrobacter strains

Summary Glycerol-fermenting anaerobes were enriched with glycerol at low and high concentrations in order to obtain strains that produce 1,3-propanediol. Six isolates were selected for more detailed characterization; four of them were identified as Citrobacter freundii, one as Klebsiella oxytoca and one as K. pneumoniae. The Citrobacter strains formed 1.3-propanediol and acetate and almost no by-products, while the Klebsiella strains produced varying amounts of ethanol in addition and accordingly less 1,3-propanediol. Enterobacterial strains of the genera Enterobacter, Klebsiella, and Citrobacter from culture collections showed similar product patterns except for one group which formed limited amounts of ethanol, but no propanediol. Seven strains were grown in pH-controlled batch cultures to determine the parameters necessary to evaluate their capacity for 1,3-propanediol production. K. pneumoniae DSM 2026 exhibited the highest final concentration (61 g/l) and the best productivity (1.7 g/l h) whereas C. freundii Zu and K2 achieved only 35 g/l and 1.4 g/l h, respectively. The Citrobacter strains on the other hand gave somewhat better yields which were very close to the theoretical optimum of 65 mol %. Offprint requests to: H. Biebl     

Reconstruction of the violacein biosynthetic pathway from Duganella sp. B2 in different heterologous hosts

Violacein is a bacteria-originated indolocarbazole pigment with potential applications due to its various bioactivities such as anti-tumor, antiviral, and antifungal activities. However, stable mass production of this pigment is difficult due to its low productivities and the instability of wild-type violacein-producing strains. In order to establish a stable and efficient production system for violacein, the violacein synthesis pathway from a new species of Duganella sp. B2 was reconstructed in different bacterial strains including Escherichia coli, Citrobacter freundii, and Enterobacter aerogenes by using different vectors. The gene cluster that encodes five enzymes involved in the violacein biosynthetic pathway was first isolated from Duganella sp. B2, and three recombinant expression vectors were constructed using the T7 promoter or the alkane-responsive promoter PalkB. Our results showed that violacein could be stably synthesized in E. coli, C. freundii, and E. aerogenes. Interestingly, we found that there were great differences between the different recombinant strains, not only in the protein expression profiles pertaining to violacein biosynthesis but also in the productivity and composition of crude violacein. Among the host strains tested, the crude violacein production by the recombinant C. freundii strain reached 1.68 g L⁻¹ in shake flask cultures, which was 4-fold higher than the highest production previously reported in flask culture by other groups. To the best of our knowledge, this is the first report on the efficient production of violacein by genetically engineered strains.     

Isolation and characterisation of bacterial strains containing enantioselective DMSO reductase activity: application to the kinetic resolution of racemic sulfoxides

The kinetic resolution of racemic sulfoxides by dimethyl sulfoxide (DMSO) reductases was investigated with a range of microorganisms. Three bacterial isolates (provisionally identified as Citrobacter braakii, Klebsiella sp. and Serratia sp.) expressing DMSO reductase activity were isolated from environmental samples by anaerobic enrichment with DMSO as terminal electron acceptor. The organisms reduced a diverse range of racemic sulfoxides to yield either residual enantiomer depending upon the strain used. C. braakii DMSO-11 exhibited wide substrate specificity that included dialkyl, diaryl and alkylaryl sulfoxides, and was unique in its ability to reduce the thiosulfinate 1,4-dihydrobenzo-2, 3-dithian-2-oxide. DMSO reductase was purified from the periplasmic fraction of C. braakii DMSO-11 and was used to demonstrate unequivocally that the DMSO reductase was responsible for enantiospecific reductive resolution of racemic sulfoxides.     

Taxonomy of Citrobacter rods found in human specimens

The aim of this study was the identification of 181 Citrobacter strains on the basis of the recently proposed taxonomic changes of Brenner. All strains were isolated from diarrhoeic patients; 124 strains were originally sent for identification to Laboratory of Enterobacteriaceae DB NIH, 57 strains was isolated in Czech Republic. Citrobacter isolates were initially identified as C. koseri (3 strains), C. amalonaticus (1 strain) or as members of the C. freundii complex (177 strains). Additionally some biochemical tests were performed. The ability to grow in medium containing KCN, lysine decarboxylase production, lactose fermentation and PYR test were examined. Strains belonging to the C. freundii complex were identified to the species level by biochemical methods on the basis of the results of Brenner, who found some tests to be useful in separating Citrobacter species. These test included citrate and acetate utilization, arginine dihydrolase and ornithine decarboxylase activities, motility, urease production, esculin hydrolysis, and acid production from sucrose, dulcitol, melibiose, raffinose and salicin. On the basis of the criteria described above, 96.6% of the strains tested could be assigned to one of the recently named species of C. freundii complex. Using biochemical tests suggested by Brenner we were able to identify Citrobacter strains members of newly recognised species. A five-test system is proposed to identify the most frequently encountered species currently residing in the C. freundii complex.     

Diarrhea-associated biofilm formed by enteroaggregative <Emphasis Type="Italic">Escherichia coli</Emphasis> and aggregative <Emphasis Type="Italic">Citrobacter freundii</Emphasis>: a consortium mediated by putative F pili

Background  

Enteroaggregative Escherichia coli (EAEC) are enteropathogenic strains identified by the aggregative adhesion (AA) pattern that share the capability to form biofilms. Citrobacter freundii is classically considered as an indigenous intestinal species that is sporadically associated with diarrhea.     

Interaction of various bacteriocins withKlebsiella edwardsii var.edwardsii

The interaction of four different bacteriocins produced byKlebsiella pneumoniae andCitrobacter freundii strains with cells ofKlebsiella edwardsii var.edwardsii has been studied. All four bacteriocins have different activity spectra. The existence of multi-tolerant and multi-receptor-negative mutants supports the hypothesis that the specific receptor sites for these bacteriocins on sensitive bacteria have some components in common.Bacteriocins S6 and S8, produced byKlebsiella pneumoniae strains inhibit protein biosynthesis. Colicin A, produced byCitrobacter freundii inhibits all macromolecular synthesis, but pre-treatment of sensitive cells with colicin A had no influence on the production of ATP by oxidative phosphorylation in cell homogenates. Bacteriocin G196, also produced byCitrobacter freundii inhibits protein and RNA synthesis, with little effect on DNA synthesis. Homogenates of cells pre-treated with bacteriocin G196, show a substantial phosphorylating activity.The authors wish to thank Dr. W. de Vries for performing P:O measurements. The skilful technical assistance of Miss E. A. Spanjaerdt Speckman and Miss W. M. C. Kapteijn is gratefully acknowledged.The investigations were supported (in part) by the Netherlands Foundation for Chemical Research (SON) with financial aid from the Netherlands Organization for the Advancement of Pure Research (ZWO).     

Structures of the O-polysaccharides of Salmonella enterica O59 and Escherichia coli O15

The O-polysaccharide of Salmonella enterica O59 was studied using sugar analysis and 2D ¹H and ¹³C NMR spectroscopy, and the following structure of the tetrasaccharide repeating unit was established:→2)-β-d-Galp-(1→3)-α-d-GlcpNAc-(1→4)-α-l-Rhap-(1→3)-β-d-GlcpNAc-(1→Accordingly, the O-antigen gene cluster of S. enterica O59 includes all genes necessary for the synthesis of this O-polysaccharide. Earlier, another structure has been reported for the O-polysaccharide of Salmonella arizonae (S. enterica IIIb) O59, which later was found to be identical to that of Citrobacter (Citrobacter braakii) O35 and, in this work, also to the O-polysaccharide of Escherichia coli O15.     

Host controlled restriction and modification of bacteriophage Mu and Mu-promoted chromosome mobilization in Citrobacter freundii

Summary Bacteriophage Mu grown on Escherichia coli K12 (Mu. K) is restricted by wild type Citrobacter freundii. In two C. freundii mutants, where the restriction of foreign F factors is absent (de Graaff and Stouthamer, 1971), the restriction for Mu. K, although at a lower level, still exists. Consequently two host specificity systems exist in C. freundii, one affecting mainly the acceptance of foreign plasmidal and chromosomal DNA and one affecting foreign DNA of bacteriophage Mu. Mu is able to lysogenize C. freundii and to induce mutations at random in its chromosome. Furthermore Mu is able to promote the mobilization of the C. freundii chromosome in strains carrying F factors. Mu promoted integration of F ts 114 lac ⁺ into the C. freundii chromosome was observed, resulting in the formation of stable Hfr strains. In this way it is possible to devise a method for chromosome transfer in other genera than E. coli to which plasmids of E. coli can be transferred, but in which no chromosome mobilization is possible because of poor DNA homology between the foreign plasmid and the host chromosome.     

Production of L-DOPA and dopamine in recombinant bacteria bearing the Vitreoscilla hemoglobin gene

Given the well-established beneficial effects of Vitreoscilla hemoglobin (VHb) on heterologous organisms, the potential of this protein for the production of L -DOPA and dopamine in two bacteria, Citrobacter freundii and Erwinia herbicola, was investigated. The constructed recombinants bearing the VHb gene (vgb⁺) had substantially higher levels of cytoplasmic L -DOPA (112 mg/L for C. freundii and 97 mg/L for E. herbicola) than their respective hosts (30.4 and 33.8 mg/L) and the vgb^– control strains (35.6 and 35.8 mg/L). Further, the vgb⁺ recombinants of C. freundii and E. herbicola had 20-fold and about two orders of magnitude higher dopamine levels than their hosts, repectively. The activity of tyrosine phenol-lyase, the enzyme converting L -tyrosine to L -DOPA, was well-correlated to cytoplasmic L -DOPA levels. As cultures aged, higher tyrosine phenol-lyase activity of the vgb⁺ strains was more apparent.     

Microbial spermidine dehydrogenase: Purification and properties of the enzyme in Pseudomonas aeruginosa and Citrobacter freundii

Spermidine dehydrogenases were purified from Pseudomonas aeruginosa and Citrobacter freundii. P. aeruginosa cells contained only one constitutive spermidine dehydrogenase. C. freundii cells contained two kinds of spermidine dehydrogenases (I and II) and both were induced in the presence of substrate, spermidine. The enzymes purified from the two strains consisted of a single polypeptide chain with a molecular weight of about 63,000. The enzymes (I and II) in C. freundii had a sharp absorbance peak at 430 nm and showed the same N-terminal amino acid sequences (Ser-Gly-Lys-Gly-Asn-). The enzymes in P. aeruginosa and C. freundii specifically oxidized spermidine and spermine in the presence of electron acceptors such as potassium ferrycyanide and 2, 6-dichlorophenolindophenol.     

Mutations in the gyrA and parC genes associated with fluoroquinolone resistance in clinical isolates of Citrobacter freundii

We determined partial sequences of the gyrA and parC genes of Citrobacter freundii type strain, and then examined 38 C. freundii clinical strains isolated from patients with urinary tract infections for the association of alterations in GyrA and ParC with susceptibility to fluoroquinolones. Our results suggest that in C. freundii DNA gyrase may be a primary target of quinolones, that an amino acid change at Thr-83 or Asp-87 in GyrA is sufficient to decrease susceptibility to fluoroquinolones, and that accumulation of changes in GyrA with the simultaneous presence of an alteration at Ser-80 or Glu-84 in ParC may be associated with the development of high-level fluoroquinolone resistance in C. freundii clinical isolates.     

Improvement of tempe fermentations by application of mixed cultures consisting of Rhizopus sp. and bacterial strains

Tempe fermentations using mixed cultures of Rhizopus oligosporus MS5, R. oryzae EN, Citrobacter freundii, and Brevibacterium epidermidis were investigated. Consumption of 150 g tempe, produced with a pure fungal mixed culture out of strains MS5 and EN, is sufficient to cover the daily requirements of niacin, vitamin K, ergosterol, and tocopherol as well as half of the daily requirement of pyridoxine, riboflavin, and biotin. Moreover, one-fourth of the recommended amount of folate is supplied. Supplementation of the fungal inoculum with C. freundii results in tempe enriched with vitamin B₁₂. Menachinone was produced as a typical bacterial vitamin K derivative. Metabolic activity of C.␣freundii led to an additional decrease of the α-galactosides stachyose and raffinose compared to pure fungal fermentations. No bacterial formation of factor 2 could be observed. Received: 31 July 1996 / Received revision: 4 October 1996 / Accepted: 14 October 1996     

Enzymatic reduction of the α, β-unsaturated carbon bond in citral

Bacteria, yeasts and filamentous fungi were screened for enantio-specific reduction of the α, β-unsaturated carbon bond in citral to produce citronellal. While a traditional aqueous screening system revealed only Zymomonas mobilis as positive, citronellal was produced in an aqueous/organic two liquid phase system by 11 of the 46 tested strains, which demonstrates the relevance of applying two-phase systems to screening strategies. Z. mobilis and Citrobacter freundii formed 1 mM citronellal in 3 h in the presence of a NADPH regenerating system and 20% (v/v) toluene. In comparison to these bacteria, the eukaryotic strains showed at least five-fold lower citral reductase activities. The bacterial strains produced preferentially the (S)-enantiomer of citronellal with e.e. values of >99% for Z. mobilis and 75% for Citrobacter freundii. In contrast the yeasts produced preferentially (R)-citronellal, i.e. Candida rugosa with an enantiomeric excess value of more than 98%. Many strains formed alcoholic by-products, viz. geraniol, nerol and citronellol. For Z. mobilis the production of these alcohols was suppressed in the presence of various organic solvents, e.g. toluene, and further decreased after EDTA addition.     

Characterization and purification of a bacteriocin from <Emphasis Type="Italic">Lactobacillus paracasei</Emphasis> subsp. <Emphasis Type="Italic">paracasei</Emphasis> BMK2005, an intestinal isolate active against multidrug-resistant pathogens

A strain of Lactobacillus paracasei subsp. paracasei BMK2005 isolated from healthy infant faeces has shown a remarkable antibacterial activity against 32 bacterial pathogenic strains of human clinical isolates. Among them, 13 strains belonging to species of Escherichia coli, Citrobacter freundii, Citrobacter diversus, Klebsiella oxytoca, Enterobacter cloacae and Pseudomonas aeruginosa were resistant to Cefotaxime (CTX) and Ceftazidime (CAZ), and 4 strains of Staphylococcus aureus were resistant to Methicillin (MRSA). This antibacterial activity was attributed to a bacteriocin designated as Paracaseicin A. It was heat-stable up to 120°C for 5 min and active within the pH range of 2–5. Its activity was lost when treated with proteases, which reveals its proteinaceous nature. This bacteriocin was successfully purified only by two steps of reversed phase chromatography. Its molecular mass, determined by mass spectrometry analysis, was 2,462.5 Da. To our knowledge, the present study is the first report on characterization and purification of a bacteriocin, produced by a L. paracasei subsp. paracasei strain exhibiting an antibacterial activity against various multidrug-resistant species of Gram-positive and Gram-negative bacteria, which reveals its potential for use in prevention or treatment of infections caused by multidrug-resistant species especially in cases of antibiotics-associated diarrhea (AAD).     

Molecular analysis of type 3 fimbrial genes from <Emphasis Type="Italic">Escherichia coli</Emphasis>, <Emphasis Type="Italic">Klebsiella</Emphasis> and <Emphasis Type="Italic">Citrobacter</Emphasis> species

Background  

Catheter-associated urinary tract infection (CAUTI) is the most common nosocomial infection in the United States and is caused by a range of uropathogens. Biofilm formation by uropathogens that cause CAUTI is often mediated by cell surface structures such as fimbriae. In this study, we characterised the genes encoding type 3 fimbriae from CAUTI strains of Escherichia coli, Klebsiella pneumoniae, Klebsiella oxytoca, Citrobacter koseri and Citrobacter freundii.     

Inhibition ofCitrobacter freundii by lactic acid, ascorbic acid, citric acid,Thymus vulgaris extract and NaCl at 31 °C and 5 °C

Citrobacter freundii has been implicated in food spoilage and food poisoning outbreaks. This study examines the effects of some compounds (e.g. citric acid, ascorbic acid, lactic acid, sodium chloride, andThymus vulgaris extract) on growth of two strains of Citrobacter freundii at 31 °C and 5 °C. At 31 °C, lactic acid (0.2%) or ascorbic acid (0.2%) alone completely inhibited growth of the tested strains, as there was 100% reduction in growth of the strains after 24 h incubation in nutrient broth containing these compounds.Thymus vulgaris extract (0.3%) reduced the growth rate (p<0.05), the percentages of inhibition after 24 h incubation were about 60% for both strains. NaCl (5%) greatly reduced growth, the percentages of inhibition were about 84% for both strains. Combination ofT. vulgaris extract (0.3%) and NaCl (4%) together completely inhibited growth ofC. freundii species tested. Ascorbic acid (0.1%) or citric acid (0.03%) did not affect growth of the strains (p>0.05), but a lag occurred before increase in number could be observed. In chicken and fish homogenates, combination of NaCl (4%) and ascorbic acid (0.1%) reduced the growth (p < 0.05) (growth inhibition was 40%). At 5 °C, lactic acid (0.1%) alone greatly reduced the growth (p<0.05). The activity of NaCl, or ascorbic acid alone against the tested strains was greatly increased (p<0.05). ForC. freundii 4, the percentage of growth inhibition after 6 days incubation in broth containing 3% NaCl or 0.1% ascorbic acid were 88% and 72%, respectively. ForC. freundii 38, the percentage of growth inhibition after 6 days incubation in broth containing these compounds were 60% and 54%, respectively.     

Effects of Varying Concentrations of Novobiocin Incorporated into Two Salmonella Plating Media on the Recovery of Four Enterobacteriaceae

Hydrogen sulfide-producing strains of salmonellae, Escherichia coli, Citrobacter freundii, and Proteus mirabilis were isolated from fresh pork sausage. All the strains produced black-centered colonies on Hektoen enteric agar (HE). On xylose lysine deoxycholate agar (XLD), C. freundii produced yellow colonies, and the strains of the other three genera formed black-centered colonies. The selectivity of HE and XLD for salmonellae was improved by the addition of novobiocin to both media. With increasing concentrations of novobiocin, the degree of growth inhibition for the four genera was less on HE than on XLD. Novobiocin concentrations of 80 μg/ml in HE and 5 μg/ml in XLD did not affect the growth or colonial morphology of salmonellae. When 80 μg of novobiocin per ml was incorporated into HE, P. mirabilis strains were not recovered, 40% of C. freundii strains failed to form black-centered colonies, and growth of E. coli strains was not affected but colonies were altered without eliminating the black centers. When novobiocin at 5 μg/ml was incorporated into XLD, colonies of P. mirabilis strains were not recovered, C. freundii formed yellow colonies, and the colonies of the H₂S-producing E. coli strains were unaffected.     

Formate dependent nitrate and nitrite reduction to ammonia by Citrobacter freundii and competition with denitrifying bacteria

Citrobacter freundii, Paracoccus denitrificans and Pseudomonas stutzeri were grown either singly or in mixed culture in anaerobic nitrate or nitrite limited chemostats with formate and/or succinate as electron donors and carbon sources. C. freundii reduced nitrate or nitrite stoichiometrically to ammonia. Maximum molar growth yields for nitrate (nitrite) were 15.3 (9.9) g/mol for C. freundii on formate with succinate as carbon source, 15.3 (9.5) g/mol for Ps. stutzeri on succinate and 32.3 (20.4) g/mol for Pa. denitrificans on succinate. The almost identical growth yields indicate that the ATP output of the anaerobic processes in the nitrate (nitrite) ammonifying organism and Ps. stutzeri are nearly the same. In mixed cultures with either Ps. stutzeri or Pa. denitrificans, C. freundii was the best competitor for nitrate. These results show that in anaerobic environments C. freundii may compete successfully with denitrifying organisms.