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Archives of Microbiology (v.188, #5)
Biological characterization of a clinical and an environmental isolate of Acanthamoeba polyphaga: analysis of relevant parameters to decode pathogenicity by Bruno da Rocha-Azevedo; Fernando Costa e Silva-Filho (pp. 441-449).
Acanthamoeba spp. consists of free-living amoebae, widespread in nature, which occasionally can cause human infections including granulomatous amoebic encephalitis and amoebic keratitis. Acanthamoeba pathogenesis is not entirely known and correlations between pathogenic potential and taxonomy are complex issues. In order to decipher the definition of a pathogenic amoeba, the objective of this work was to decipher the definition of pathogenic amoeba by characterizing two isolates of Acanthamoeba polyphaga obtained from different origins (a keratitis patient and freshwater), looking for differences among them. The clinical isolate grew faster in Peptone-yeast extract-glucose (PYG) medium, transformed more rapidly from a trophozoite to cyst and exhibited increased cytopathic effect on cultured cells. Morphological differences were also noted, since freshwater amoebae presented more acanthopodia than the clinical isolate. Moreover, actin labeling demonstrated that microfilament organization varies between isolates, with the presence of locomotory structures as lobopodia and lamellipodia in the keratitis isolate, which were less adherent on plastic. Zymography demonstrated that the keratitis isolates presented higher proteolytic activity and also were more able to invade collagen matrices. Altogether, we conclude that a group of stable physiological characteristics exist in Acanthamoeba that can be related to pathogenicity.
Keywords: Acanthamoeba ; Pathogenic protozoa; Free-living amoebae; Isolates characterization; Cytopathic effect; Proteases; Adhesion; Collagen invasion
Molecular characterization of Pseudomonas sp. LDC-5 involved in accumulation of poly 3-hydroxybutyrate and medium-chain-length poly 3-hydroxyalkanoates by Kabilan Sujatha; Ayyasamy Mahalakshmi; Rajaiah Shenbagarathai (pp. 451-462).
Polyhydroxyalkanoates (PHAs) are biological polyesters, of which, Short-Chain-Length-Medium-Chain-Length (SCL-MCL) PHA copolymers are important because of their wide range of applications. The present study focused on molecular characterization of Pseudomonas sp. LDC-5 that is identified as SCL-MCL producer. Phase contrast, fluorescent and electron microscopic observation confirmed the presence of PHA granules in Pseudomonas sp. LDC-5. PCR analysis indicated the presence of expected amplicon for SCL phaC gene (∼500 bp), MCL phaC1 with phaZ (∼1.3), and phaC2 with phaZ (∼1.5 kb). Sequence analysis of the PHA synthase gene of Pseudomonas sp. LDC-5 revealed significant differences in phaC1 and phaC2 which were further confirmed by recombinant studies. Recombinant Escherichia coli harboring the partial phaC1 gene was able to accumulate PHA, whereas E. coli with phaC2 did not accumulate PHA as verified by fold analysis, immunoblotting, Gas Chromatography (GC), Differential scanning calorimetry (DSC), and FTIR studies. The predicted theoretical three-dimensional structure revealed that PhaC1 is consistent with α/β hydrolase fold. Monomer composition showed the presence of monomer ranging from C4 to C12: 1 when glucose and sodium octanoate fed as the carbon source. DSC revealed melting temperature peak at 153.12°C and glass transition (Tg) peaks at −0.37°C. Thermogravimetric analysis revealed that the polymer was stable up to 276°C. Fourier Transform Infrared Spectroscopy (FT-IR) spectral analysis showed the PHA specific wave number at 1,739.67 and 1,161.07 cm−1. The potential of Pseudomonas sp. LDC-5 and its properties are discussed.
Keywords: Pseudomonas sp. LDC-5; SCL-MCL PHA; α/β hydrolase fold; Lipase box; Fluorescent microscopy; phaC operon; PHA synthase
Genetic and proteomic analyses of CO utilization by Methanosarcina acetivorans by Michael Rother; Ellen Oelgeschläger; William W. Metcalf (pp. 463-472).
Methanosarcina acetivorans, a member of the methanogenic archaea, can grow with carbon monoxide (CO) as the sole energy source and generates, unlike other methanogens, substantial amounts of acetate and formate in addition to methane. Phenotypic analyses of mutant strains lacking the cooS1F operon and the cooS2 gene suggest that the monofunctional carbon monoxide dehydrogenase (CODH) system contributes to, but is not required for, carboxidotrophic growth of M. acetivorans. Further, qualitative proteomic analyses confirm a recent report (Lessner et al., Proc Natl Acad Sci USA, 103:17921–17926, 2006) in showing that the bifunctional CODH/acetyl-CoA synthase (ACS) system, two enzymes involved in CO2-reduction, and a peculiar protein homologous to both corrinoid proteins and methyltransferases are synthesized at elevated levels in response to CO; however, the finding that the latter protein is also abundant when trimethylamine serves as growth substrate questions its proposed involvement in the reduction of methyl-groups to methane. Potential catabolic mechanisms and metabolic adaptations employed by M. acetivorans to effectively utilize CO are discussed.
Keywords: Methanosarcina acetivorans ; Carbon monoxide; Carbon monoxide dehydrogenase
Activation of Helicobacter pylori inorganic pyrophosphatase and the importance of Cys16 in thermostability, enzyme activation and quaternary structure by Mon-Juan Lee; Haimei Huang; Wei Lin; Ray-Rong Yang; Chien-Liang Liu; Chung-Yu Huang (pp. 473-482).
The inorganic pyrophosphatase from the human pathogen Helicobacter pylori (HpPPase) is a family I PPase. It is a homohexamer consisting of identical 20-kDa subunits. Hydrolysis of inorganic pyrophosphate (PPi) by HpPPase relied on the presence of magnesium and followed Michaelis–Menten kinetics, with k cat being 344 s−1 and K m being 83 μM at pH 8.0, which was the optimal pH for catalysis. HpPPase was activated by both thiol and non-thiol reductants, distinct from the previously suggested inactivation/reactivation process involving formation and breakage of disulfide bonds. Substitution of Cys16 of HpPPase, which was neither located at the active site nor evolutionarily conserved, resulted in a loss of 50% activity and a reduction in sensitivity to reductants and oxidized glutathione. In addition, the C16S replacement caused a considerable disruption in thermostability, which exceeded that resulted from active-site mutations such as Y140F HpPPase and those of Escherichia coli. Although Cys16 was not located at the subunit interface of the hexameric HpPPase, sedimentation analysis results suggested that the C16S substitution destabilized HpPPase through impairing trimer–trimer interactions. This study provided the first evidences that the single cysteine residue of HpPPase was involved in enzyme activation, thermostability, and stabilization of quaternary structure.
Keywords: Inorganic pyrophosphatase (PPase); Helicobacter pylori ; Site-directed mutagenesis; Thermostability; Reductants
Molecular characterization of plant growth promoting rhizobacteria that enhance peroxidase and phenylalanine ammonia-lyase activities in chile (Capsicum annuum L.) and tomato (Lycopersicon esculentum Mill.) by Alok Sharma; Ashutosh Pathak; Manvika Sahgal; Jean-Marie Meyer; Victor Wray; Bhavdish N. Johri (pp. 483-494).
Pythium and Phytophthora species are associated with damping-off diseases in vegetable nurseries and reduce seedling stand and yield. In this study, bacterial isolates were selected on the basis of in vitro antagonism potential to inhibit mycelial growth of damping-off pathogens along with plant growth properties for field assessment in wet and winter seasons. We demonstrate efficacy of bacterial isolates to protect chile and tomato plants under natural vegetable nursery and artificially created pathogen-infested (Pythium and Phytophthora spp.) nursery conditions. After 21 days of sowing, chile and tomato plants were harvested and analysed for peroxidase and phenylalanine ammonia-lyase activities. Pseudomonas sp. strains FQP PB-3, FQA PB-3 and GRP3 were most effective in increasing shoot length (P > 0.05%) in both artificial and natural field sites. For example, Pseudomonas sp. FQA PB-3 treatment increased shoot length by 40% in the artificial Pythium 4746 infested nursery site in chile plants in the wet season. The bacterial treatments significantly increased the activity of peroxidase and phenylalanine ammonia-lyase in chile and tomato plant tissues, which are well known as indicators of an active lignification process. Thus, we conclude that treatment with potential bacterial plant growth promoting agents help plants against pathogen invasion by modulating plant peroxidase and phenylalanine ammonia-lyase activities.
Keywords: Plant stress enzymes; ARDRA; Damping-off; Phytopathogen; Rhizobacteria
Diverse rhizobia that nodulate two species of Kummerowia in China by Dong Xu Lin; Chao Xin Man; En Tao Wang; Wen Xin Chen (pp. 495-507).
A total of 63 bacterial strains were isolated from root nodules of Kummerowia striata and K. stipulacea grown in different geographic regions of China. These bacteria could be divided into fast-growing (FG) rhizobia and slow-growing (SG) rhizobia according to their growth rate. Genetic diversity and taxonomic relationships among these rhizobia were revealed by PCR-based 16 S rDNA RFLP and sequencing, 16 S-IGS RFLP, SDS-PAGE of whole cell soluble proteins, BOX-PCR and symbiotic gene (nifH/nodC) analyses. The symbiotic FG strains were mainly isolated from temperate regions and they were identified as four genomic species in Rhizobium and Sinorhizobium meliloti based on the consensus of grouping results. The SG strains were classified as five genomic species within Bradyrhizobium and they were mainly isolated fron the subtropic and tropical regions. The phylogenetic analyses of nifH and nodC genes showed relationships similar to that of 16 S rDNA but the symbiotic genes of Bradyrhizobium strains isolated from Kummerowia were distinct from those isolated from Arachis and soybean. These results offered evidence for rhizobial biogeography and demonstrated that the Kummerowia-nodulating ability might have evolved independently in different regions in association with distinctive genomic species of rhizobia.
Keywords: Diversity; Phylogeny; Rhizobia; Kummerowia ; Biogeography
Transport of Na+ and K+ by an antiporter-related subunit from the Escherichia coli NADH dehydrogenase I produced in Saccharomyces cerevisiae by Anja C. Gemperli; Christiane Schaffitzel; Claude Jakob; Julia Steuber (pp. 509-521).
The NADH dehydrogenase I from Escherichia coli is a bacterial homolog of the mitochondrial complex I which translocates Na+ rather than H+. To elucidate the mechanism of Na+ transport, the C-terminally truncated NuoL subunit (NuoLN) which is related to Na+/H+ antiporters was expressed as a protein A fusion protein (ProtA–NuoLN) in the yeast Saccharomyces cerevisiae which lacks an endogenous complex I. The fusion protein inserted into membranes from the endoplasmatic reticulum (ER), as confirmed by differential centrifugation and Western analysis. Membrane vesicles containing ProtA–NuoLN catalyzed the uptake of Na+ and K+ at rates which were significantly higher than uptake by the control vesicles under identical conditions, demonstrating that ProtA–NuoLN translocated Na+ and K+ independently from other complex I subunits. Na+ transport by ProtA–NuoLN was inhibited by EIPA (5-(N-ethyl-N-isopropyl)-amiloride) which specifically reacts with Na+/H+ antiporters. The cation selectivity and function of the NuoL subunit as a transporter module of the NADH dehydrogenase complex is discussed.
Keywords: Bacterial respiration; Membrane protein expression; NADH dehydrogenase; Na+ transporter; Cation antiporter
Identification of a novel potential antigen for early-phase serodiagnosis of leptospirosis by Fernanda O. Neves; Patricia A. E. Abreu; Silvio A. Vasconcellos; Zenáide M. de Morais; Eliete C. Romero; Ana L. T. O. Nascimento (pp. 523-532).
This study examined four genes encoding for predicted membrane proteins selected from the genome sequences of Leptospira interrogans. Genes were cloned and the proteins expressed in E. coli. Immunoblotting analysis of the recombinants with sera from early and convalescent phases of a leptospirosis patient showed that two proteins, namely Lp29 and Lp49, were reactive with serum from both phases of the illness. These data were further confirmed in enzyme-linked immunosorbent assay using sera from both phases of seventeen confirmed leptospirosis specimens, suggesting that these proteins are presented to the host immune system during infection. In the early phase, anti-Lp29 IgM was detected in all sera when microscopic agglutination tests (MAT), the reference method for diagnosis of leptospirosis, were negative. The gene encoding Lp49 is conserved among five tested leptospiral pathogenic serovars, while Lp29 is present in serovars that are predominant in urban settings. These recombinant antigens might be valuable for serodiagnosis of both phases of leptospirosis.
Keywords: Leptospira interrogans ; Leptospirosis; Cloning and expression; Diagnostic
Study of methanol-induced phenotypic changes in a novel strain of Acinetobacter lwoffii by Anuradha Ghosh; Abhineet Goyal; Rakesh K. Jain (pp. 533-539).
A Gram-negative bacterial strain designated LS2 isolated from Lahaul–Spiti valley of North India was shown to produce pink pigment while utilizing methanol as sole source of carbon and energy. Interestingly, pigment production was inducible in nature since the organism did not produce any pigment when grown on other carbon sources. Based on phenotypic and phylogenetic characterization the non-pigmented methylotroph was identified as a novel strain of Acinetobacter lwoffii MTCC 8288 (DQ144736). By means of spectral and mass analyses the pigment was characterized as bacterioruberin-like carotenoid molecule. Here, the carotenoid pigment may form an important part of the antioxidant defense mechanism against oxidative stress imparted by methanol. The methanol utilization pathway in strain LS2 was deciphered by showing the presence of functional methanol dehydrogenase and formaldehyde dehydrogenase genes. In addition, to investigate methanol induced physiological changes, comparative fatty acid profile was analysed and distinctive qualitative as well as quantitative differences in fatty acid content were observed. Therefore, we suggest that strain LS2 exhibiting such unique phenotypic property should be assigned a taxonomic position other than the pigmented and non-pigmented methylotrophs.
Keywords: Acinetobacter lwoffii ; Non-pigmented methylotroph; Methanol utilization; Carotenoid pigment; Fatty acid analysis
The Streptomyces coelicolor dnaK operon contains a second promoter driving the expression of the negative regulator hspR at physiological temperature by Paola Salerno; Sandra Marineo; Anna Maria Puglia (pp. 541-546).
HspR (heat shock protein regulator) acts as a negative regulator of different genes in many bacteria. In Streptomyces coelicolor hspR gene is part and the transcriptional repressor of the dnaK operon which encodes the DnaK, GrpE, DnaJ chaperone machines and HspR itself. Our experiments led us to the discovery of a second promoter, internal to dnaK operon, located upstream hspR gene. Transcription from this promoter was detected at 30°C indicating that hspR could play a key physiological role.
Keywords: Streptomyces coelicolor ; Heat shock response; hspR
New species in the genus Francisella (Gammaproteobacteria; Francisellaceae); Francisella piscicida sp. nov. isolated from cod (Gadus morhua) by Karl F. Ottem; Are Nylund; Egil Karlsbakk; Alice Friis-Møller; Bjørn Krossøy; Dag Knappskog (pp. 547-550).
A Francisella strain, GM2212, previously isolated from moribund farmed Atlantic cod (Gadus morhua) in Norway, is closely related to Francisella philomiragia among Francisella spp. according to its complete 16S rDNA, 16S-23S intergenic spacer, 23S rDNA, 23S–5S intergenic spacer, 5S rDNA, FopA, lipoprotein TUL4 (LpnA), malate dehydrogenase and hypothetical lipoprotein (LpnB) sequences. A comparison between GM2212 and the type strain of Francisella philomiragia were performed by DNA–DNA hybridization and fatty acid analysis. The DNA–DNA hybridization showed a 70% similarity. The fatty acid analysis showed only minor differences between the Francisella isolates. Due to the inconclusive result from the DNA–DNA hybridisation, major emphasis concerning the status of this isolate is made on previously published molecular, phenotypic and biochemical characters. All characteristics taken together support the establishment of GM2212 as a novel species, for which the name Francisella piscicida sp. nov. is proposed (=CNCM I-3511T = DSM 18777T = LMG registration number not yet available).
Keywords: Francisella piscicida sp. nov.; DNA–DNA hybridization; Fatty acids