NMR solution structure of KP-TerB, a tellurite-resistance protein from Klebsiella pneumoniae

Klebsiella pneumoniae (KP), a Gram-negative bacterium, is a common cause of hospital-acquired bacterial infections worldwide. Tellurium (Te) compounds, although relatively rare in the environment, have a long history as antimicrobial and therapeutic agents. In bacteria, tellurite (TeO(3) (-2)) resistance is conferred by the ter (Te(r)) operon (terZABCDEF). Here, on the basis of 2593 restraints derived from NMR analysis, we report the NMR structure of TerB protein (151 amino acids) of KP (KP-TerB), which is mainly composed of seven alpha-helices and a 3(10) helix, with helices II to V apparently forming a four-helix bundle. The ensemble of 20 NMR structures was well-defined, with a RMSD of 0.32 +/- 0.06 A for backbone atoms and 1.11 +/- 0.07 A for heavy atoms, respectively. A unique property of the KP-TerB structure is that the positively and negatively charged clusters are formed by the N-terminal positively and C-terminal negatively charged residues, respectively. To the best of our knowledge, the protein sequence and structures of KP-TerB are unique.     

Whole genome sequence of Klebsiella pneumoniae U25, a hypermucoviscous,multidrug resistant,biofilm producing isolate from India

Klebsiella pneumoniae U25 is a multidrug resistant strain isolated from a tertiary care hospital in Chennai, India. Here, we report the complete annotated genome sequence of strain U25 obtained using PacBio RSII. This is the first report of the whole genome of K. pneumoniaespecies from Chennai. It consists of a single circular chromosome of size 5,491,870-bp and two plasmids of size 211,813 and 172,619-bp. The genes associated with multidrug resistance were identified. The chromosome of U25 was found to have eight antibiotic resistant genes [blaOXA-1,blaSHV-28, aac(6’)1b-cr,catB3, oqxAB, dfrA1]. The plasmid pMGRU25-001 was found to have only one resistant gene (catA1) while plasmid pMGRU25-002 had 20 resistant genes [strAB, aadA1,aac(6’)-Ib, aac(3)-IId,sul1,2, blaTEM-1A,1B,blaOXA-9, blaCTX-M-15,blaSHV-11, cmlA1, erm(B),mph(A)]. A mutation in the porin OmpK36 was identified which is likely to be associated with the intermediate resistance to carbapenems in the absence of carbapenemase genes. U25 is one of the few K. pneumoniaestrains to harbour clustered regularly interspaced short palindromic repeats (CRISPR) systems. Two CRISPR arrays corresponding to Cas3 family helicase were identified in the genome. When compared to K. pneumoniaeNTUHK2044, a transposase gene InsH of IS5-13 was found inserted.     

Contemplating 3-Hydroxypropionic Acid Biosynthesis in Klebsiella pneumoniae

3-Hydroxypropionic acid (3-HP) is a commercially valuable platform chemical from which an array of C₃ compounds can be generated. Klebsiella pneumoniae has been considered a promising species for biological production of 3-HP. Despite a wealth of reports related to 3-HP biosynthesis in K. pneumoniae, its commercialization is still in infancy. The major hurdle hindering 3-HP overproduction lies in the poor understanding of glycerol dissimilation in K. pneumoniae. To surmount this problem, this review aims to portray a picture of 3-HP biosynthesis, involving 3-HP-synthesizing strains, biochemical attributes, metabolic pathways and key enzymes. Inspired by the state-of-the-art advances in metabolic engineering and synthetic biology, here we advocate protocols for overproducing 3-HP in K. pneumoniae. These protocols range from cofactor regeneration, alleviation of metabolite toxicity, genome editing, remodeling of transport system, to carbon flux partition via logic gate. The feasibility for these protocols was also discussed.

Electronic supplementary material

The online version of this article (doi:10.1007/s12088-015-0513-0) contains supplementary material, which is available to authorized users.     

Gene Arrangements in Expression Vector Affect 3-Hydroxypropionic Acid Production in Klebsiella pneumoniae

Biosynthesis of 3-hydroxypropionic acid (3-HP) typically involves two sequential reactions catalyzed by glycerol dehydratase (DhaB) and aldehyde dehydrogenase (AldH). Although plasmid-dependent over-expression of the two enzymes is common, systematic investigation of gene arrangement in vector has not been reported. Here we show that gene arrangements have a noticeable influence on 3-HP production. Using Klebsiella pneumoniae as a host, three AldH-coding genes: ald4 from Saccharomyces cerevisiae, aldh from Escherichia coli, and puuC from host K. pneumoniae, were respectively ligated to dhaB. The recombinant Kp/pET-pk-ald4-dhaB (Kp refers to as K. pneumoniae, pk is a native promoter) produced the highest yield of 3-HP in comparison to both Kp/pET-pk-dhaB-ald4 and Kp/pET-pk-dhaB-pk-ald4, suggesting that the preferential expression of AldH can increase 3-HP production. Additionally, when different AldH-coding genes were respectively ligated downstream of dhaB, the recombinant Kp/pET-pk-dhaB-puuC produced more 3-HP than that by Kp/pET-pk-dhaB-aldh or Kp/pET-pk-dhaB-ald4, implying the intrinsic compatibility of native gene puuC with its host. These findings indicate the applicability of native AldH-coding gene and provide insights into strategies for metabolic engineering of multiple genes.     

Efficient production of ethanol from crude glycerol by a Klebsiella pneumoniae mutant strain

A mutant strain of Klebsiella pneumoniae, termed GEM167, was obtained by γ irradiation, in which glycerol metabolism was dramatically affected on exposure to γ rays. Levels of metabolites of the glycerol reductive pathway, 1,3-propanediol (1,3-PD) and 3-hydroxypropionic acid (3-HP), were decreased in the GEM167 strain compared to a control strain, whereas the levels of metabolites derived from the oxidative pathway, 2,3-butanediol (2,3-BD), ethanol, lactate, and succinate, were increased. Notably, ethanol production from glycerol was greatly enhanced upon fermentation by the mutant strain, to a maximum production level of 21.5 g/l, with a productivity of 0.93 g/l/h. Ethanol production level was further improved to 25.0 g/l upon overexpression of Zymomonas mobilispdc and adhII genes encoding pyruvate decarboxylase (Pdc) and aldehyde dehydrogenase (Adh), respectively in the mutant strain GEM167.     

Molecular typing of Klebsiella pneumoniae isolates from public hospitals in Recife, Brazil

Thirty nosocomial isolates of Klebsiella pneumoniae, collected from three public hospitals in Recife, Brazil, between 1999 and 2000, were analysed in order to determine their epidemiological relatedness and genetic characteristics. The isolates were characterised by biotyping, antibiotyping, protein analysis, plasmid profile and random amplified polymorphic DNA (RAPD). The majority of the clinical isolates were resistant to multiple antibiotics, in particular to beta-lactams, and 30% were found to be ESBLs producers. RAPD proved to be the most effective technique in discriminating unrelated K. pneumoniae isolates. It was confirmed by the highly genetic similarity found among related isolates from an hospital outbreak. We conclude that K. pneumoniae infections in Recife has been caused by a variety of bacterial genotypes. This is the first report that revealed the subtypes of K. pneumoniae in Brazil by plasmid analysis and RAPD.     

High Prevalence of Exfoliative Toxins Among Carrier Isolates of Staphylococcus aureus from Healthy Individuals from Various Communities in Chennai, South India

Staphylococcus aureus causes infections both in community and hospital settings, nasal carriage is the important source of these infections. A total of 103 carrier isolates of S. aureus from 352 asymptomatic individuals were screened for methicillin-resistant S. aureus (MRSA) and exfoliative toxins (A, B and D) by two sets of multiplex PCRs. The overall nasal carriage of MRSA was found to be 13/352 (3.7 %), of which 4 were found to be positive for Panton valentine leucocidin (PVL). Twelve (11.65 %) strains were found to carry exfoliative toxins and belonged to one of the following spa types t159, t209 and t1515. High prevalence of exfoliative toxins, pvl and MRSA pose a major threat to public health, since the isolates were from the healthy in various community settings.     

Simultaneous production of 3-hydroxypropionic acid and 1,3-propanediol from glycerol by a recombinant strain of Klebsiella pneumoniae

In this study, an aldehyde dehydrogenase (ALDH) was over-expressed in Klebsiella pneumoniae for simultaneous production of 3-hydroxypropionic acid (3-HP) and 1,3-propanediol (1,3-PDO). Various genes encoding ALDH were cloned and expressed in K. pneumoniae, and expression of Escherichia colialdH resulted in the highest 3-HP titer in anaerobic cultures in shake flasks. Anaerobic fed-batch culture of this recombinant strain was further performed in a 5-L reactor. The 3-HP concentration and yield reached 24.4 g/L and 0.18 mol/mol glycerol, respectively, and at the same time 1,3-PDO achieved 49.3 g/L with a yield of 0.43 mol/mol in 24 h. The overall yield of 3-HP plus 1,3-PDO was 0.61 mol/mol. Over-expression of the E. coli AldH also reduced the yields of by-products except for lactate. This study demonstrated the possibility of simultaneous production of 3-HP and 1,3-PDO by K. pneumoniae under anaerobic conditions without supply of vitamin B₁₂.     

An optimized method for suicide vector-based allelic exchange in Klebsiella pneumoniae

Klebsiella pneumoniae is an important and versatile bacterium that can be found in diverse environments and is also a frequent cause of human infections. Limited data exists on the mechanisms of interaction between K. pneumoniae and the human host and of adaptations to other environments. Coupled with the high genetic diversity of this species, these factors highlight the necessity for substantial further K. pneumoniae-focused molecular genetics studies.In this report we describe a simple and efficient experimental protocol for suicide vector-based allelic exchange in K. pneumoniae. The protocol has been validated by mutating multiple loci in four distinct K. pneumoniae strains, including highly capsulated and/or multi-antibiotic resistant clinical isolates. Three key enhancements are reported:(1) Use of pDS132-derived conjugative plasmids carrying improved cloning sites, (2) Performance of sacB counterselection at 25 °C as opposed to higher temperatures, and (3) Exploitation of Flp-recombinase-mediated deletion of FRT (Flp recombinase target) flanked resistance cassettes to allow for reiterative manipulations with a single selectable marker. This study also highlights a problem that may be encountered when the aacC1 gentamicin resistance marker is used in K. pneumoniae and suggests alternative markers.The protocol developed in this study will help investigate the plethora of uncharacterized genes present in the K. pneumoniae pan-genome and shed further light upon clinically and industrially important phenotypes observed in this ubiquitous species.     

Prevalence and Clinical Manifestations of Malaria in Aligarh,India

Malaria is one of the most widespread infectious diseases of tropical countries with an estimated 207 million cases globally. In India, there are endemic pockets of this disease, including Aligarh. Hundreds of Plasmodium falciparum and P. vivax cases with severe pathological conditions are recorded every year in this district. The aim of this study is to find out changes in liver enzymes and kidney markers. Specific diagnosis for P. falciparum and P. vivax was made by microscopic examination of Giemsa stained slides. Clinical symptoms were observed in both of these infections. Liver enzymes, such as AST, ALT, and ALP, and kidney function markers, such as creatinine and urea, were estimated by standard biochemical techniques. In Aligarh district, P. vivax, P. falciparum, and mixed infections were 64%, 34%, and 2%, respectively. In case of P. falciparum infection, the incidences of anemia, splenomegaly, renal failure, jaundice, and neurological sequelae were higher compared to those in P. vivax infection. Recrudescence and relapse rates were 18% and 20% in P. falciparum and P. vivax infections, respectively. Liver dysfunctions and renal failures were more common in P. falciparum patients, particularly in elderly patients. Artesunate derivatives must, therefore, be introduced for the treatment of P. falciparum as they resist to chloroquine as well as sulfadoxine-pyrimethamine combinations.     

Structural and Mechanistic Studies on Klebsiella pneumoniae 2-Oxo-4-hydroxy-4-carboxy-5-ureidoimidazoline Decarboxylase

The stereospecific oxidative degradation of uric acid to (S)-allantoin was recently shown to proceed via three enzymatic steps. The final conversion is a decarboxylation of the unstable intermediate 2-oxo-4-hydroxy-4-carboxy-5-ureidoimidazoline (OHCU) and is catalyzed by OHCU decarboxylase. Here we present the structures of Klebsiella pneumoniae OHCU decarboxylase in unliganded form and with bound allantoin. These structures provide evidence that ligand binding organizes the active site residues for catalysis. Modeling of the substrate and intermediates provides additional support for this hypothesis. In addition we characterize the steady state kinetics of this enzyme and report the first OHCU decarboxylase inhibitor, allopurinol, a structural isomer of hypoxanthine. This molecule is a competitive inhibitor of K. pneumoniae OHCU decarboxylase with a K_i of 30 ± 2 μm. Circular dichroism measurements confirm structural observations that this inhibitor disrupts the necessary organization of the active site. Our structural and biochemical studies also provide further insights into the mechanism of catalysis of OHCU decarboxylation.     

1H and 13C NMR characterization and secondary structure of the K2 polysaccharide of Klebsiella pneumoniae strain 52145

The complete (1)H and (13)C NMR characterization of the tetrasaccharide repeating unit from the K2 polysaccharide of Klebsiella pneumoniae strain 52145 is reported. [chemical structure] In addition a model for its secondary structure was suggested on the basis of dynamic and molecular calculations.     

Production of 1,3-propanediol by Klebsiella pneumoniae from glycerol broth

Broth containing 152 g glycerol l⁻¹ from Candida krusei culture was converted to 1,3-propanediol by Klebsiella pneumoniae. Residual glucose in the broth promoted growth of K. pneumoniae while acetate was inhibitory. After desalination treatment of glycerol broth by electrodialysis, the acetate in the broth was removed. A fed-batch culture with electrodialytically pretreated broth as␣substrate was developed giving 53 g 1,3- propanediol l⁻¹ with a yield of 0.41 g g⁻¹ glycerol and a productivity of 0.94 g l⁻¹ h⁻¹.     

Outbreak of carbapenem-resistant Klebsiella pneumoniae: two-year epidemiologic follow-up in a tertiary hospital

This study describes a carbapenem-resistant Klebsiella pneumoniae (CRKP) outbreak that occurred from October 2008-December 2010. Polymerase chain reaction assays were performed to detect the bla_KPC gene and molecular typing was performed using pulsed-field gel electrophoresis (PFGE). There were 33 CRKP infections; PFGE revealed five genotypes: genotype A in five (15%), B in 18 (55%), C in eight (24%) and two unique profiles. Genotype B was disseminated in all hospital units and belonged to the same clone identified in 11 different hospitals in the state of São Paulo. Sixteen (48%) patients died. Seven isolates (21%) were resistant to polymyxin B and 45% were resistant to tigecycline and amikacin.     

Production of 3-hydroxypropionic acid through propionaldehyde dehydrogenase PduP mediated biosynthetic pathway in Klebsiella pneumoniae

The pduP gene encodes a propionaldehyde dehydrogenase (PduP) was investigated for the role in 3-hydroxypropionic acid (3-HP) glycerol metabolism in Klebsiella pneumoniae. The enzyme assay showed that cell extracts from a pduP mutant strain lacked measurable dehydrogenase activity. Additionally, the mutant strain accumulated the cytotoxic intermediate metabolite 3-hydroxypropionaldehyde (3-HPA), causing both cell death and a lower final 3-HP titer. Ectopic expression of pduP restored normal cell growth to mutant. The enzymatic property of recombinant protein from Escherichia coli was examined, exhibiting a broad substrate specificity, being active on 3-HPA. The present work is thus the first to demonstrate the role of PduP in glycerol metabolism and biosynthesis of 3-HP.     

Expansion of the known Klebsiella pneumoniae species gene pool by characterization of novel alien DNA islands integrated into tmRNA gene sites

Klebsiella pneumoniae is an important bacterial pathogen of man that is commonly associated with opportunistic and hospital-associated infections. Increasing levels of multiple-antibiotic resistance associated with this species pose a major emerging clinical problem. This organism also occurs naturally in other diverse environments, including the soil. Consistent with its varied lifestyle and membership of the Enterobacteriaceae family, K. pneumoniae genomes exhibit highly plastic architecture comprising a core genome backbone interspersed with numerous and varied alien genomic islands. In this study the size of the presently known K. pneumoniae pan-genome gene pool was estimated through analysis of complete sequences of three chromosomes and 31 plasmids belonging to K. pneumoniae strains. In addition, using a PCR-based strategy the genomic content of eight tRNA/tmRNA gene sites that serve as DNA insertion hotspots were investigated in 28 diverse environmental and clinical strains of K. pneumoniae. Sequencing and characterization of five newly identified horizontally-acquired tmRNA-associated islands further expanded the archived K. pneumoniae gene pool to a total of 7648 unique gene members. Large-scale investigation of the content of tRNA/tmRNA hotspots will be useful to identify and/or survey accessory sequences dispersed amongst hundreds to thousands of members of many key bacterial species.     

Metabolism of acrylonitrile by Klebsiella pneumoniae

A gram-negative rod-shaped bacterium capable of utilizing acrylonitrile as the sole source of nitrogen was isolated from industrial sewage and identified as Klebsiella pneumoniae. The isolate was capable of utilizing aliphatic nitriles containing 1 to 5 carbon atoms or benzonitrile as the sole source of nitrogen and either acetamide or propionamide as the sole source of both carbon and nitrogen. Gas chromatographic and mass spectral analyses of culture filtrates indicated that K. pneumoniae was capable of hydrolyzing 6.15 mmol of acrylonitrile to 5.15 mmol of acrylamide within 24 h. The acrylamide was hydrolyzed to 1.0 mmol of acrylic acid within 72 h. Another metabolite of acrylonitrile metabolism was ammonia, which reached a maximum concentration of 3.69 mM within 48 h. Nitrile hydratase and amidase, the two hydrolytic enzymes responsible for the sequential metabolism of nitrile compounds, were induced by acrylonitrile. The optimum temperature for nitrile hydratase activity was 55°C and that for amidase was 40°C; both enzymes had pH optima of 8.0.Abbreviations PBM phosphate buffered medium - GC gas chromatography - GC/MS gas chromatography/mass spectrometry     

Growth and Starvation of a Strain of Klebsiella pneumoniae Isolated from a Brazilian Oil Formation

Summary The changes that occur in cell size and shape during experimental starvation and resuscitation of bacteria may find practical application in the biotechnological processes used in microbial enhanced oil-recovery (MEOR), in in situ bioremediation and in the creation of biobarriers. In the work described here, the aim was to observe certain aspects of the response of a strain of Klebsiella pneumoniae, isolated from oil producer wells belonging to PETROBRAS (National Oil Company of Brazil), to starvation and growth under various culture conditions. Cells of K. pneumoniae were taken from an exponentially-growing culture, washed and suspended in phosphate-buffered saline solution, without nutrients, where they remained for 91 days. Aliquots were withdrawn periodically, to perform viable and total cell counts and measure the optical density and cell dimensions. The starved cells were resuspended in sodium citrate medium, where they recuperated and exhibited a typical growth curve. The results indicate that this strain is a viable option for future trials on the transport and growth of microorganisms inside porous media, aimed at possible applications in MEOR.     

Characterisation of mutations in the Klebsiella pneumoniae nitrogen fixation regulatory gene nifL which impair oxygen regulation

The nifL gene product of Klebsiella pneumoniae inhibits the activity of the positive activator protein NifA in response to increased levels either of fixed nitrogen or of oxygen in the medium. In order to demonstrate that the responses to these two effectors are discrete we have subjected nifL to hydroxylamine mutagenesis and isolated nifL mutants that are impaired in their ability to respond to oxygen but not to fixed nitrogen. Two such mutations were sequenced and shown to be single base pair changes located in different parts of nifL. The amino acid sequence of NifL shows limited homology to the histidine protein kinases which comprise the sensing component of bacterial two-component regulatory systems. In the light of the location of one of the oxygen-insensitive mutations (Leu294Phe) we have reassessed this homology and we suggest that the Gln273-Leu317 region of NifL may facilitate interactions between NifL and NifA.Abbreviations X-gal 5-bromo-4-chloro-3-indolyl--D-galactopy-ranoside - USAs upstream activator sequences