<Emphasis Type="Italic">R</Emphasis>-acetoin accumulation and dissimilation in <Emphasis Type="Italic">Klebsiella pneumoniae</Emphasis>

Klebsiella pneumoniae is a 2,3-butanediol producer, and R-acetoin is an intermediate of 2,3-butanediol production. R-acetoin accumulation and dissimilation in K. pneumoniae was studied here. A budC mutant, which has lost 2,3-butanediol dehydrogenase activity, accumulated high levels of R-acetoin in culture broth. However, after glucose was exhausted, the accumulated R-acetoin could be reused by the cells as a carbon source. Acetoin dehydrogenase enzyme system, encoded by acoABCD, was responsible for R-acetoin dissimilation. acoABCD mutants lost the ability to grow on acetoin as the sole carbon source, and the acetoin accumulated could not be dissimilated. However, in the presence of another carbon source, the acetoin accumulated in broth of acoABCD mutants was converted to 2,3-butanediol. Parameters of R-acetoin production by budC mutants were optimized in batch culture. Aerobic culture and mildly acidic conditions (pH 6–6.5) favored R-acetoin accumulation. At the optimized conditions, in fed-batch fermentation, 62.3 g/L R-acetoin was produced by budC and acoABCD double mutant in 57 h culture, with an optical purity of 98.0 %, and a substrate conversion ratio of 28.7 %.     

A simple method for extracting C-phycocyanin from <Emphasis Type="Italic">Spirulina platensis</Emphasis> using <Emphasis Type="Italic">Klebsiella pneumoniae</Emphasis>

C-phycocyanin (C-PC) was extracted from fresh Spirulina platensis by deploying a species of non-pathogenic nitrogen-fixing bacteria, namely, Klebsiella pneumoniae. The algal slurry was neither washed nor centrifuged; the bacterial culture was poured into the slurry, the vessel sealed, and crude C-PC extracted after about 24 h. The extraction was clean and efficient, and the purity and concentration of C-PC proved to be of adequate quality.     

Inactivation of <Emphasis Type="Italic">dhaD</Emphasis> and <Emphasis Type="Italic">dhaK</Emphasis> abolishes by-product accumulation during 1,3-propanediol production in <Emphasis Type="Italic">Klebsiella pneumoniae</Emphasis>

1,3-Propanediol (1,3-PD) can be used for the industrial synthesis of a variety of compounds, including polyesters, polyethers, and polyurethanes. 1,3-PD is generated from petrochemical and microbial sources. 1,3-Propanediol is a typical product of glycerol fermentation, while acetate, lactate, 2,3-butanediol, and ethanol also accumulate during the process. Substrate and product inhibition limit the final concentration of 1,3-propanediol in the fermentation broth. It is impossible to increase the yield of 1,3-propanediol by using the traditional whole-cell fermentation process. In this study, dhaD and dhaK, the genes for glycerol dehydrogenase and dihydroxyacetone kinase, respectively, were inactivated by homologous recombination in Klebsiella pneumoniae. The dhaD/dhaK double mutant (designated TC100), selected from 5,000 single or double cross homologous recombination mutants, was confirmed as a double cross by using polymerase chain reaction. Analysis of the cell-free supernatant with high-performance liquid chromatography revealed elimination of lactate and 2,3-butanediol, as well as ethanol accumulation in TC100, compared with the wild-type strain. Furthermore, 1,3-propanediol productivity was increased in the TC100 strain expressing glycerol dehydratase and 1,3-PDO dehydrogenase regulated by the arabinose P_BAD promoter. The genetic engineering and medium formulation approaches used here should aid in the separation of 1,3-propanediol from lactate, 2,3-butanediol, and ethanol and lead to increased production of 1,3-propanediol in Klebsiella pneumoniae.     

Amoxicillin and specific bacteriophage can be used together for eradication of biofilm of <Emphasis Type="Italic">Klebsiella pneumoniae</Emphasis> B5055

Despite the efficacy of antibiotics as well as bacteriophages in treatment of bacterial infections, their role in treatment of biofilm associated infections is still under consideration especially in case of older biofilms. Here, efficacy of bacteriophage alone or in combination with amoxicillin, for eradication of biofilm of Klebsiella pneumoniae B5055 has been assessed. Planktonic cells as well as biofilm of K. pneumoniae B5055 grown in 96-well microtiter plates were exposed to bacteriophage and amoxicillin at various Multiplicity of Infections (MoIs) as well as at three different antibiotic concentrations (512, 256 and 128 μg/ml), respectively. After exposure to 256 μg/ml (MIC) of amoxicillin, bacterial load of planktonic culture as well as 1-day-old biofilm was reduced by a log factor of 4.1 ± 0.31 (P = 0.008) and 1.24 ± 0.27 (P < 0.05), respectively but reduction in the bacterial load of mature biofilm (8-day-old) was insignificant (P = 0.23). When 8-day-old biofilm was exposed to higher antibiotic concentration (512 μg/ml) or phage alone (MoI = 0.01) a log reduction of 2.97 ± 0.11 (P = 0.182) and 3.51 ± 0.19 (P = 0.073), respectively was observed. While on exposing to a combination of both the amoxicillin and phage, a significant reduction (P < 0.01) in bacterial load of the biofilm was seen. Hence, when antibiotic was used in combination with specific bacteriophage a greater destruction of the biofilm structure suggested that the phages could be used successfully along with antibiotic therapy. An added advantage of the combination therapy would be its ability to check formation of resistant mutants that otherwise develop easily upon using phage or antibiotic alone.     

Non-capsulated mutants of a chemical-producing <Emphasis Type="Italic">Klebsiella pneumoniae</Emphasis> strain

Objectives

To investigate the outcomes of capsule lost on cell transformation efficiency and chemicals (1,3-propanediol, 2,3-butanediol, and 2-ketogluconic acid) production by Klebsiella pneumoniae.

Results

The cps gene cluster showed low sequence homology with pathogenic strains. The wza is a highly conserved gene in the cps cluster that encodes an outer membrane protein. A non-capsulated mutant was constructed by deletion of wza. Phenotype studies demonstrated that non-capsulated cells were less buoyant and easy to sediment. The transformation efficiency of the non-capsulated mutant reached 6.4 × 10⁵ CFU μg^?1 DNA, which is 10 times higher than that of the wild strain. 52.2 g 1,3-propanediol L^?1, 30.7 g 2,3-butanediol L^?1, and 175.9 g 2-ketogluconic acid L^?1 were produced by non-capsulated mutants, which were 10–40% lower compared to wild strain. Furthermore, viscosities of the three fermentation broths decreased to approximately 1.3 cP from the range of 1.8–2.2 cP.

Conclusions

Non-capsulated K. pneumoniae mutants should allay concerns regarding biological safety, improve transformation efficiency, lower viscosity, and subsequently ameliorate the financial burden of the downstream process of chemicals production.

     

Enhanced 2,3-butanediol production by <Emphasis Type="Italic">Klebsiella pneumoniae</Emphasis> SDM

Enhanced 2,3-butanediol (BD) production was carried out by Klebsiella pneumoniae SDM. The nutritional requirements for BD production by K. pneumoniae SDM were optimized statistically in shake flask fermentations. Corn steep liquor powder and (NH₄)₂HPO₄ were identified as the most significant factors by the two-level Plackett–Burman design. Steepest ascent experiments were applied to approach the optimal region of the two factors and a central composite design was employed to determine their optimal levels. The optimal medium was used to perform fed-batch fermentations with K. pneumoniae SDM. BD production was then studied in a 5-l bioreactor applying different fed-batch strategies, including pulse fed batch, constant feed rate fed batch, constant residual glucose concentration fed batch, and exponential fed batch. The maximum BD concentration of 150 g/l at 38 h with a diol productivity of 4.21 g/l h was obtained by the constant residual glucose concentration feeding strategy. To the best of our knowledge, these results were new records on BD fermentation. Cuiqing Ma and Ailong Wang contributed equally to this work.     

In Vitro Management of Hospital <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> Biofilm Using Indigenous T7-Like Lytic Phage

Pseudomonas aeruginosa, a human pathogen capable of forming biofilm and contaminating medical settings, is responsible for 65% mortality in the hospitals all over the world. This study was undertaken to isolate lytic phages against biofilm forming Ps. aeruginosa hospital isolates and to use them for in vitro management of biofilms in the microtiter plate. Multidrug resistant strains of Ps. aeruginosa were isolated from the hospital environment in and around Pimpri-Chinchwad, Maharashtra by standard microbiological methods. Lytic phages against these strains were isolated from the Pavana river water by double agar layer plaque assay method. A wide host range phage bacterial virus Ps. aeruginosa phage (BVPaP-3) was selected. Electron microscopy revealed that BVPaP-3 phage is a T7-like phage and is a relative of phage species gh-1. A phage at MOI-0.001 could prevent biofilm formation by Ps. aeruginosa hospital strain-6(HS6) on the pegs within 24 h. It could also disperse pre-formed biofilms of all hospital isolates (HS1–HS6) on the pegs within 24 h. Dispersion of biofilm was studied by monitoring log percent reduction in cfu and log percent increase in pfu of respective bacterium and phage on the peg as well as in the well. Scanning electron microscopy confirmed that phage BVPaP-3 indeed causes biofilm reduction and bacterial cell killing. Laboratory studies prove that BVPaP-3 is a highly efficient phage in preventing and dispersing biofilms of Ps. aeruginosa. Phage BVPaP-3 can be used as biological disinfectant to control biofilm problem in medical devices.     

Characterization of 1,3-propanediol oxidoreductase (DhaT) from <Emphasis Type="Italic">Klebsiella pneumoniae</Emphasis> J2B

1,3-propanediol oxidoreductase (DhaT) of Klebsiella pneumoniae converts 3-hydroxypropionaldehyde (3-HPA) to 1,3-propanediol (1,3-PD) during microbial production of 1,3-PD from glycerol. In this study, DhaT from newly isolated K. pneumoniae J2B was cloned, expressed, purified, and studied for its kinetic properties. It showed, on its physiological substrate 3-HPA, higher activity than similar aldehydes such as acetaldehyde, propionaldehyde and butyraldehyde. The turnover numbers (k _cat , 1/s) were estimated as 59.4 for the forward reaction (3-HPA to 1,3-PD at pH 7.0) and 10.0 for the reverse reaction (1,3-PD to 3-HPA at pH 9.0). The Michaelis constants (K _m , mM) were 0.77 (for 3-HPA) and 0.03 (for NADH) for the forward reaction (at pH 7.0), and 7.44 (for 1,3-PD) and 0.23 (for NAD⁺) for the reverse reaction (at pH 9.0). Between these forward and reverse reactions, the optimum temperature and pH were significantly different (37°C and 7.0 vs. 55°C and 9.0, respectively). These results indicate that, under physiological conditions, DhaT mostly catalyzes the forward reaction. The enzyme was seriously inhibited by heavy metal ions such as Ag⁺ and Hg²⁺. DhaT was highly unstable when incubated with its own substrate 3-HPA, indicating the necessity of enhancing its stability for improved 1,3-PD production from glycerol.     

Deletion of <Emphasis Type="Italic">ldh</Emphasis>A and <Emphasis Type="Italic">ald</Emphasis>H genes in <Emphasis Type="Italic">Klebsiella</Emphasis><Emphasis Type="Italic">pneumoniae</Emphasis> to enhance 1,3-propanediol production

Objectives

To improve 1,3-propanediol (1,3-PD) production and reduce byproduct concentration during the fermentation of Klebsiella pneumonia.

Results

Klebsiella. pneumonia 2-1ΔldhA, K. pneumonia 2-1ΔaldH and K. pneumonia 2-1ΔldhAΔaldH mutant strains were obtained through deletion of the ldhA gene encoding lactate dehydrogenase required for lactate synthesis and the aldH gene encoding acetaldehyde dehydrogenase involved in the synthesis of ethanol. After fed-batch fermentation, the production of 1,3-PD from glycerol was enhanced and the concentrations of byproducts were reduced compared with the original strain K. pneumonia 2-1. The maximum yields of 1,3-PD were 85.7, 82.5 and 87.5 g/l in the respective mutant strains.

Conclusion

Deletion of either aldH or ldhA promoted 1,3-PD production in K. pneumonia.

     

Mapping phosphoproteins in <Emphasis Type="Italic">Mycoplasma genitalium</Emphasis> and <Emphasis Type="Italic">Mycoplasma pneumoniae</Emphasis>

Background  

Little is known regarding the extent or targets of phosphorylation in mycoplasmas, yet in many other bacterial species phosphorylation is known to play an important role in signaling and regulation of cellular processes. To determine the prevalence of phosphorylation in mycoplasmas, we examined the CHAPS-soluble protein fractions of Mycoplasma genitalium and Mycoplasma pneumoniae by two-dimensional gel electrophoresis (2-DE), using a combination of Pro-Q Diamond phosphoprotein stain and ³³P labeling. Protein spots that were positive for phosphorylation were identified by peptide mass fingerprinting using MALDI-TOF-TOF mass spectrometry.     

Volatile metabolic diversity of <Emphasis Type="Italic">Klebsiella pneumoniae</Emphasis> in nutrient-replete conditions

Introduction

Microorganisms catabolize carbon-containing compounds in their environment during growth, releasing a subset of metabolic byproducts as volatile compounds. However, the relationship between growth media and the production of volatile compounds has been largely unexplored to-date.

Objectives

To assess the core and media-specific components of the Klebsiella pneumoniae volatile metabolome via growth in four in vitro culture media.

Methods

Headspace volatiles produced by cultures of K. pneumoniae after growth to stationary phase in four rich media (brain heart infusion broth, lysogeny broth, Mueller-Hinton broth, and tryptic soy broth) were analyzed using comprehensive two-dimensional gas chromatography time-of-flight mass spectrometry (GC×GC-TOFMS). Differences in the composition of headspace volatiles as a function of growth media were assessed using hierarchical clustering analysis (HCA) and principal component analysis (PCA).

Results

A total of 365 volatile compounds were associated with the growth of K. pneumoniae across all media, of which 36 (10%) were common to all growth media, and 148 (41%) were specific to a single medium. In addition, utilizing all K. pneumoniae-associated volatile compounds, strains clustered as a function of growth media, demonstrating the importance of media in determining the metabolic profile of this organism.

Conclusion

K. pneumoniae produces a core suite of volatile compounds across all growth media studied, although the volatile metabolic signature of this organism is fundamentally media-dependent.

     

<Emphasis Type="Italic">Ty</Emphasis>1<Emphasis Type="Italic">/copia</Emphasis>- and <Emphasis Type="Italic">Ty</Emphasis>3<Emphasis Type="Italic">/gypsy</Emphasis>-like DNA sequences in <Emphasis Type="Italic">Helianthus </Emphasis>species

Two repeated DNA sequences isolated from a partial genomic DNA library of Helianthus annuus, p HaS13 and p HaS211, were shown to represent portions of the int gene of a Ty3 /gypsy retroelement and of the RNase-Hgene of a Ty1 /copia retroelement, respectively. Southern blotting patterns obtained by hybridizing the two probes to BglII- or DraI-digested genomic DNA from different Helianthus species showed p HaS13 and p HaS211 were parts of dispersed repeats at least 8 and 7 kb in length, respectively, that were conserved in all species studied. Comparable hybridization patterns were obtained in all species with p HaS13. By contrast, the patterns obtained by hybridizing p HaS211 clearly differentiated annual species from perennials. The frequencies of p HaS13- and p HaS211-related sequences in different species were 4.3x10(4)-1.3x10(5) copies and 9.9x10(2)-8.1x10(3) copies per picogram of DNA, respectively. The frequency of p HaS13-related sequences varied widely within annual species, while no significant difference was observed among perennial species. Conversely, the frequency variation of p HaS211-related sequences was as large within annual species as within perennials. Sequences of both families were found to be dispersed along the length of all chromosomes in all species studied. However, Ty3 /gypsy-like sequences were localized preferentially at the centromeric regions, whereas Ty1/ copia-like sequences were less represented or absent around the centromeres and plentiful at the chromosome ends. These findings suggest that the two sequence families played a role in Helianthusgenome evolution and species divergence, evolved independently in the same genomic backgrounds and in annual or perennial species, and acquired different possible functions in the host genomes.     

<Emphasis Type="Italic">Klebsiella pneumoniae</Emphasis> antibiotic resistance identified by atomic force microscopy

In the last decade the detection of the resistance of bacteria to antibiotics treatment, developed by different kind of bacteria, is becoming a huge problem. We hereby present a different approach to the current problem of detection of bacteria resistance to antibiotics. Our aims were to use the atomic force microscopy (AFM) to investigate bacteria morphological changes in response to antibiotics treatment and explore the possibility of reducing the time required to obtain information on their resistance. In particular, we studied Klebsiella pneumoniae bacteria provided by the Lavagna Hospital ASL4 Liguria (Italy), where there are cases linked with antibiotics resistance of the Klebsiella pneumoniae. By comparing AFM images of bacteria strains treated with different antibiotics is possible to identify unambiguously the Klebsiella pneumoniae strains resistant to antibiotics. In fact, the analysis of the AFM images of the antibiotic-sensitive bacteria shows clearly the presence of morphological alterations in the cell wall. While in the case of the antibiotic-resistant bacteria morphological alterations are not present. This approach is based on an easy and potentially rapid AFM analysis.     

Decrease of 3-hydroxypropionaldehyde accumulation in 1,3-propanediol production by over-expressing <Emphasis Type="Italic">dha</Emphasis>T gene in <Emphasis Type="Italic">Klebsiella pneumoniae</Emphasis> TUAC01

Glycerol can be biologically converted to 1,3-propanediol, a key raw material required for the synthesis of polytrimethylene terephthalate and other polyester fibers. In 1,3-propanediol synthesis pathway, 3-hydroxypropionaldehyde (3-HPA) was an inhibitory intermediary metabolite. The accumulation of 3-HPA in broth would cause an irreversible cessation of the fermentation process. With the object of reducing 3-HPA level in the fermentation broth, dhaT gene which encodes 1,3-propanediol oxidoreductase (PDOR) was cloned and over expressed in 1,3-propanediol producing bacterium Klebsiella pneumoniae TUAC01. dhaT gene was linked downstream of the ptac promoter in an expressing vector pDK6 to form plasmid pDK-dhaT. The newly formed pDK-dhaT was transformed to K. pneumoniae TUAC01. Under the inducement of IPTG, PDOR was over-expressed when the constructed strain was cultured on an LB medium or a fermentation medium. A 5 L scale-up fermentation experiment was done to test the 3-HPA accumulation in broth, with the initial substrate glycerol 30 g/L; the peak levels of 3-HPA in broth were 7.55 and 1.49 mmol/L for control host strain and the constructed strain, respectively. In 50 g/L initial glycerol experiment, the peak level of 3-HPA in broth was 12.57 and 2.02 mmol/l for the control host strain and the constructed strain, respectively. Thus the fermentation cessation caused by the toxicity of 3-HPA was alleviated in the constructed strain.     

Existence of<Emphasis Type="Italic">Sinorhizobium meliloti</Emphasis> genomic fragment hybridizing with the<Emphasis Type="Italic">nifM</Emphasis> gene of<Emphasis Type="Italic">Klebsiella pneumoniae</Emphasis>

A novel finding that genomic restriction fragments of symbiotic nitrogen fixer S. meliloti hybridized with nifM gene probe of the free-living diazotroph Klebsiella pneumoniae is reported. When SmaI endonuclease was used to digest S. meliloti DNA, a unique hybridizing band was obtained.     

Antagonistic Activity of Lactic Acid Bacteria <Emphasis Type="Italic">Lactobacillus</Emphasis> spp. against Clinical Isolates of <Emphasis Type="Italic">Klebsiella pneumoniae</Emphasis>

The screening of three strains of lactic acid bacteria identified as Lactobacillus rhamnosus, Lactobacillus reuteri, and Lactobacillus helveticus showed significant antagonistic activity against Klebsiella pneumoniae strains characterized by multiple antibiotic resistance. Lactobacilli cocultivated with the Klebsiella strains inhibited their growth 20 to 86% on the first and second days, respectively. Exoproteome analysis of L. rhamnosus cocultivated with K. pneumoniae revealed the induction of peptidoglycan hydrolases, including extracellular lytic transglycosylases, family II (MltA), and endopeptidases capable of disrupting the peptidoglycan bacterial cell wall.     

Pig farm environment as a source of beta-lactamase or AmpC-producing <Emphasis Type="Italic">Klebsiella pneumoniae</Emphasis> and <Emphasis Type="Italic">Escherichia coli</Emphasis>

The present study was undertaken to detect the occurrence of beta-lactamase-/AmpC-producing Klebsiella and Escherichia coli in healthy pigs, feed, drinking water, and pen floor or surface soil. The study also intended to detect the clonal relationship between the environmental and porcine isolates to confirm the route of transmission. Rectal swabs and environmental samples were collected from apparently healthy pigs kept in organized or backyard farms in India. The pigs had no history of antibiotic intake. Production of phenotypical beta-lactamase, associated genes, and class I integron gene was detected in E. coli and Klebsiella isolates. The phylogenetic relationship among the isolates was established on the basis of Random amplification of polymorphic DNA banding pattern. Beta-lactamase-producing Klebsiella were isolated from healthy pigs (20.0%), pen floor swabs/surface soil swabs (14.0%), and drinking water (100%). Escherichia coli isolated from healthy pigs (14.4%), pen floor/surface soil (8.0%), and drinking water (33.3%) were detected as beta-lactamase producers. Majority of beta-lactamase-producing isolates possessed bla_CTX-M-9. Further, 35 (81%) Klebsiella and all the E. coli isolates were detected as AmpC beta-lactamase ACBL producers and possessed bla_AmpC. Sixteen beta-lactamase-producing Klebsiella (37.20%) and 13 E. coli (86.67%) possessed class I integron. Few resistant isolates from environmental sources (surface soil swab and drinking water) and the studied pigs were detected within the same cluster of the dendrogram representing their similarities. The study indicated about the possible role of contaminated environment as a source of beta-lactamase/AmpC-producing Klebsiella and E. coli in pigs.