Medicharla V. Jagannadham

Sphingobacterium antarcticus sp. nov., a psychrotrophic bacterium from the soils of Schirmacher oasis, Antarctica

Two pure cultures of bacteria isolated from soil samples collected in Schirmacher Oasis, Antarctica, conformed to the definition of the genus Sphingobacterium. They differed from all of the known species of Sphingobacterium in being psychrotrophic. The G+C contents of the DNA of the two strains were found to be 39.3 and 40.3 mol%, and DNA-DNA hybridization studies indicated 7% homology with S. multivorum and S. spiritivorum. The name Sphingobacterium antarcticus sp. nov. is proposed for the two Antarctic strains. The type strain is 4BY (MTCC 675), and it has been deposited with the Microbial Type Culture Collection, Institute of Microbial Technology, Chandigarh, India.

EVpedia: a community web portal for extracellular vesicles research

MOTIVATION Extracellular vesicles (EVs) are spherical bilayered proteolipids, harboring various bioactive molecules. Due to the complexity of the vesicular nomenclatures and components, online searches for EV-related publications and vesicular components are currently challenging. RESULTS We present an improved version of EVpedia, a public database for EVs research. This community web portal contains a database of publications and vesicular components, identification of orthologous vesicular components, bioinformatic tools and a personalized function. EVpedia includes 6879 publications, 172 080 vesicular components from 263 high-throughput datasets, and has been accessed more than 65 000 times from more than 750 cities. In addition, about 350 members from 73 international research groups have participated in developing EVpedia. This free web-based database might serve as a useful resource to stimulate the emerging field of EV research. AVAILABILITY AND IMPLEMENTATION The web site was implemented in PHP, Java, MySQL and Apache, and is freely available at http://evpedia.info.

Exoribonuclease R Interacts with Endoribonuclease E and an RNA Helicase in the Psychrotrophic Bacterium Pseudomonas syringae Lz4W

Endoribonuclease E, a key enzyme involved in RNA decay and processing in bacteria, organizes a protein complex called degradosome. In Escherichia coli, Rhodobacter capsulatus, and Streptomyces coelicolor, RNase E interacts with the phosphate-dependent exoribonuclease polynucleotide phosphorylase, DEAD-box helicase(s), and additional factors in an RNA-degrading complex. To characterize the degradosome of the psychrotrophic bacterium Pseudomonas syringae Lz4W, RNase E was enriched by cation exchange chromatography and fractionation in a glycerol density gradient. Most surprisingly, the hydrolytic exoribonuclease RNase R was found to co-purify with RNase E. Co-immunoprecipitation and Ni2+-affinity pull-down experiments confirmed the specific interaction between RNase R and RNase E. Additionally, the DEAD-box helicase RhlE was identified as part of this protein complex. Fractions comprising the three proteins showed RNase E and RNase R activity and efficiently degraded a synthetic stem-loop containing RNA in the presence of ATP. The unexpected association of RNase R with RNase E and RhlE in an RNA-degrading complex indicates that the cold-adapted P. syringae has a degradosome of novel structure. The identification of RNase R instead of polynucleotide phosphorylase in this complex underlines the importance of the interaction between endo- and exoribonucleases for the bacterial RNA metabolism. The physical association of RNase E with an exoribonuclease and an RNA helicase apparently is a common theme in the composition of bacterial RNA-degrading complexes.

Steroid-induced perturbations of membranes and its relevance to sperm acrosome reaction

The interaction of progesterone, 17-alpha-hydroxyprogesterone, testosterone and estradiol with membrane vesicles prepared from phosphatidylserine (PS), from the total lipids of human and hamster spermatozoa, from the lipids of hamster spermatozoal plasma and acrosomal membrane and with the native membranes of hamster spermatozoa have been investigated by 90 degrees light scattering and fluorescence spectroscopy. The results indicate that progesterone decreases the fluidity of membranes, aggregates membrane vesicles, induces fusion of membrane vesicles and also renders them permeable to hydrophilic molecules like carboxyfluorescein. But, testosterone and estradiol at the same concentration had very little effect on membrane fluidity, membrane aggregation, fusion and leakage. The above membrane perturbing activities of the steroids is discussed in light of the recent findings that progesterone induces acrosome reaction in human and hamster spermatozoa [11,18].

Biogenesis and multifaceted roles of outer membrane vesicles from Gram-negative bacteria

Outer membrane vesicles (OMVs) released from Gram-negative bacteria consist of lipids, proteins, lipopolysaccharides and other molecules. OMVs are associated with several biological functions such as horizontal gene transfer, intracellular and intercellular communication, transfer of contents to host cells, and eliciting an immune response in host cells. Although hypotheses have been made concerning the mechanism of biogenesis of these vesicles, research on OMV formation is far from complete. The roles of outer membrane components, bacterial quorum sensing molecules and some specific proteins in OMV biogenesis have been studied. This review discusses the different models that have been proposed for OMV biogenesis, along with details of the biological functions of OMVs and the likely scope of future research.

Low-temperature-induced changes in composition and fluidity of lipopolysaccharides in the antarctic psychrotrophic bacterium Pseudomonas syringae.

The Antarctic psychrotrophic bacterium Pseudomonas syringae was more sensitive to polymyxin B at a lower (4 degrees C) temperature of growth than at a higher (22 degrees C) temperature. The amount of hydroxy fatty acids in the lipopolysaccharides (LPS) also increased at the lower temperature. These changes correlated with the increase in fluidity of the hydrophobic phase of lipopolysaccharide aggregates in vitro.

Antibiotic Resistance of Bacteria

Antibiotic resistance of bacteria and other microorganisms is one of the most serious and grievous challenges of the twenty-first century. The life-saving drugs, which held a great deal of promises during the 1940s to eradicate all the infectious life-threatening diseases in the world, have ceased to work, because of the increasing emergence of microbial strains invulnerable to them. Many of the previously efficacious antibiotics are no longer usable because of widespread occurrence of multiresistant microbial strains. Lately, discovery of new antibiotics is failing to keep pace with the emergence of (multi)resistance of pathogenic and also environmental bacterial strains. Consequently, the prospect of chemotherapy looks bleak. The trepidation that we might be pushed back to a situation analogous to the preantibiotic era, when no chemotherapeutic agent was available to contain and combat deadly bacterial infections, does not appear to be an overblown imagination. Based on this backdrop, this special issue appears to be an aptly undertaken and well-timed endeavour to address this global problem. The articles contributed by investigators from various research laboratories with different scientific backgrounds have not only portrayed the width of the problem but also displayed some silver lining in the management of the looming crisis. Rapid detection of the profile of resistance is essential for timely application of the right antibiotic to a patient. H. Frickmann et al. summarize the efficacy and limitations of various molecular and mass spectrometric methods for the detection of resistance. The omnipresent nature of the resistant organisms is revealed in a number of articles. F. B. Atique and Md. M. R. Khalil report on the occurrence of antibiotic resistance among bacteria (predominantly skin commensal coagulase-negative staphylococci) isolated from allogenic bone samples for grafting, collected from different hospitals of Bangladesh. Food materials are believed to serve as a vehicle for transmission of resistance. This issue is addressed by F. S. Dehkordi et al. who report on the genotype and resistance-profile of Helicobacter pylori isolated from vegetables and salad samples, picked up from groceries and supermarket in a province of Iran. The high similarity in the genotype pattern of the isolates obtained from vegetables and humans indicates transmission. A. B. Florez et al. reveal tetracycline and erythromycin-resistant bacteria and genes conferring resistance to these antibiotics in 10 Spanish and 10 Italian samples of commercial cheese. P. Krupa et al. report on the population structure (based on spa typing) of oxacillin-resistant Staphylococcus aureus isolated from nasal swabs of pigs, collected from two slaughter houses of Poland. Some meat samples bought from the shops were also included into their studies. D. De Vito et al. characterize multidrug-resistant clinical isolates of Salmonella typhimurium for resistance genes in an area of southern Italy by pulsotyping and phage typing. C. Zhang et al. report on the resistant phenotype and genotype of Streptococcus suis serotype 2, isolated from 62 clinically healthy sows and 34 diseased pigs reared in different farms of China. Antibiotic resistance in the nosocomial isolates is a matter of serious concern. F. Lombardi et al. look into the molecular epidemiology of carbapenemase-producing strains of Klebsiella pneumoniae isolated from the surgery unit at a cardiovascular centre of Italy. D. Ojdana et al. demonstrate the ability of an E. coli strain obtained from a hospital of Poland to produce carbapenemase enzymes and also the presence of genes responsible for the production of carbapenemases and other β-lactamases. Extended-spectrum-β-lactamase (ESBL) is a bacterial enzyme having the ability to hydrolyse even the third-generation cephalosporins and aztreonam. Besides Klebsiella pneumoniae some strains of Escherichia coli are also known to produce this enzyme. This is indicated by M. S. Rezai et al. who performed genotyping of ESBL-producing strains of E. coli, obtained from a paediatric hospital of north Iran. The authors also show the association of ESBL-positive E. coli strains with resistance to various other antimicrobials. Occurrence of ESBL-producing Enterobacteriaceae in iceberg lettuce obtained from the retail market of Rochester (US) is described by N. Bhutani et al. A wide spectrum of diseases is caused by the virulent strains of ESBL-positive isolates of E. coli. Regional difference in the prevalence of virulence genes in 432 phenotypically ESBL-positive patient-isolates of E. coli (obtained from the Baltic Sea region) is shown by J. Lillo et al. Keeping in mind the tremendous challenge posed by drug-resistant tuberculosis, a number of relevant articles are included in this collection. The susceptibility profile of M. tuberculosis isolates to various antitubercular antibiotics varies significantly depending on the test system as revealed by Z. Mei et al. They have also shown that changes in bacterial susceptibility are further caused by mixed infection with particular genotypes of M. tuberculosis strains. Resistance-profile of 100 strains of M. tuberculosis, isolated from patients in northeast Iran, is reported by A. T. Sani et al. Occurrence of nontuberculosis Mycobacterium, in 25 out of 125 patients (20%) surveyed, underscores the need of proper diagnosis before the onset of chemotherapy. Discovery of new drugs and strategies to circumvent antibiotic resistance is the need of the hour to contain the problem. N. Jafari et al. report on the isolation of an antibiotic-producing strain of a soil Actinomycetes belonging to the genus Pseudonocardia. The antibacterial compound produced by it is effective against Staphylococcus aureus. They have also purified and partially characterized this compound. R. D. Wojtyczka et al. demonstrate high antibacterial activity of two new quinoline derivatives of a structure of 3-thioacyl 1-methyl 4-arylaminoquinolinium salts against some nosocomial strains of staphylococci in both planktonic and biofilm form. In view of the widespread nature of the problem caused by inefficacy of the antibiotics produced by fermentation and chemical synthesis, it is necessary to tap alternative sources (e.g., plant kingdom) for novel antibiotics. P. Del Serrone et al. demonstrate antibacterial activity of Neem seed oil (Azadirachta indica A. Juss.) against enteropathogenic strains of E. coli and indicate that some of the ciprofloxacin-resistant isolates lost their virulence following treatment with Neem seed oil. Antimicrobial peptides are considered potential candidates for the management of multidrug-resistant infections. M. Singh and K. Mukhopadhyay evaluate the antimicrobial potential of an anti-inflammatory neuropeptide whereas C. Chen et al. report on the efficacy of recombinant lysostaphin against methicillin-resistant S. aureus (MRSA) in a mouse model. Widespread use of carbapenems is associated with emergence of resistance. The polymyxin antibiotic colistin is not used at present because of its nephrotoxicity. H.-J. Tang et al., however, demonstrate the efficacy of a combination of colistin and imipenem against carbapenem-resistant Klebsiella pneumoniae. Bacteriophages could be suitable alternatives for antibiotics, which currently have lost efficacy because of the emergence of resistant strains. N. Shivshetty et al. demonstrate the potential of a bacteriophage isolated from sewage to protect diabetic mice against Pseudomonas aeruginosa-induced bacteremia. Reversal of bacterial resistance to antibiotics is essential to restore the efficacy of the existing antimicrobials. C. Santiago et al. claim to achieve an increase in susceptibility of a MRSA strain to ampicillin when it was combined with a plant extract. A number of computerized models have been developed during the recent past to assist the physicians with the necessary information to enable prescription of the right antibiotic in the right moment. M. Rodriguez-Maresca et al. report on the efficacy of a new electronic device based on laboratory data on the most probable susceptibility profile of pathogens responsible for infections and also on local epidemiology.

Molecular characterization and functional analysis of outer membrane vesicles from the antarctic bacterium Pseudomonas syringae suggest a possible response to environmental conditions.

Outer membrane vesicles (OMVs) of Gram-negative bacteria form an important aspect of bacterial physiology as they are involved in various functions essential for their survival. The OMVs of the Antarctic bacterium Pseudomonas syringae Lz4W were isolated, and the proteins and lipids they contain were identified. The matrix-assisted laser desorption/ionization time of flight (MALDI-TOF/TOF) analysis revealed that phosphatidylethanolamines and phosphatidylglycerols are the main lipid components. The proteins of these vesicles were identified by separating them by one-dimensional gel electrophoresis and liquid chromatography coupled to electrospray ionization tandem mass spectrometry (ESI-MS/MS). They are composed of outer membrane and periplasmic proteins according to the subcellular localization predictions by Psortb v.3 and Cello V2.5. The functional annotation and gene ontology of these proteins provided hints for various functions attributed to OMVs and suggested a potential mechanism to respond to the extracellular environmental changes. The OMVs were found to protect the producer organism against the membrane active antibiotics colistin and melittin but not from streptomycin. The 1-N-phenylnapthylamine (NPN)-uptake assay revealed that the OMVs protect the bacterium from membrane active antibiotics by scavenging them and also showed that membrane and protein packing of the OMVs was similar to the parent bacterium. The sequestering depends on the composition and organization of lipids and proteins in the OMVs.

Identification of proteins from membrane preparations by a combination of MALDI TOF-TOF and LC-coupled linear ion trap MS analysis of an Antarctic bacterium Pseudomonas syringae Lz4W, a strain with unsequenced genome.

Multidimensional protein identification technology helps in identifying a large number of proteins with ESI by sequencing several peptides with MS/MS methods. When ionization and separation of different hydrophobic and hydrophilic peptides in a single process are difficult, a combination of LC‐coupled linear ion trap MS and MALDI TOF/TOF can be used for identification of proteins as shown in the present study. We have used this combinational approach to identify membrane proteins of the Antarctic bacterium Pseudomonas syringae Lz4W, which are separated by SDS gel electrophoresis. Although the genome of P. syringae Lz4W has not been sequenced, the known genome sequences of mesophilic Pseudomonas species have been used for the identification of the proteins. Broadly, many membrane proteins, proteins with a wide range of molecular weight and pI including some integral membrane proteins could be identified using this procedure. Some of the identified proteins are involved in low temperature adaptation.

Identification of outer membrane proteins from an Antarctic bacterium Pseudomonas syringae Lz4W

Subcellular fractionation of proteins is a preferred method of choice for detection and identification of proteins from complex mixtures such as bacterial cells. To characterize the membrane proteins of the Antarctic bacterium Pseudomonas syringae Lz4W, the membrane fractions were prepared using three different methods, namely Triton X-100 solubilization, sucrose density gradient, and carbonate extraction methods. The proteins were separated on one-dimensional polyacrylamide gels and analyzed using a combination of liquid chromatography-coupled electrospray ionization-MS. The membrane proteins that were prepared by carbonate extraction were separated on two-dimensional PAGE in different pI ranges using the detergent 2% amidosulfobetaine (ASB). The proteins were then subjected to matrix-assisted laser desorption ionization-time-of-flight/time-of-flight for analysis and identification. Because the genome sequence of P. syringae Lz4W is not known, the proteins were identified by using the relevant sequence databases of the Pseudomonas sp available at National Centre for Biotechnology Information (NCBI). The sequence identification of some tryptic peptides were validated by de novo sequencing and others by chemical modification and mass spectrometry. The peptide sequences of P. syringae Lz4W were then matched with the sequences of the peptides from different Pseudomonas sp. by similarity search of the proteins from different species using clustal W2 program. Thus by using a combination of the methods, we have been able to identify large number of proteins of this bacterial strain, which include most of the outer membrane proteins.