Forgot your password?
New user?
New Window Opens on the Secret Life of Microbes: Scientists Develop First Microbial Profiles of Ecosystems
CAS announces the release of SciFinder Scholar for Mac OS X
Press Releases
Press Releases
| Check out our New Publishers' Select for Free Articles |
Archives of Microbiology (v.177, #1)
No Title by Frederic Gich; Jesús Garcia-Gil; Jörg Overmann (pp. 1-10).
The phylogenetic diversity of green nonsulfur bacteria in nine stratified freshwater lakes was investigated. A set of oligonucleotide primers was developed that permitted the selective amplification of 16S rRNA gene sequences of this group. Subsequently, amplification products were separated by denaturing gradient gel electrophoresis (DGGE) and sequenced, which yielded a total of 19 novel sequence types. Ten of the sequences were related to those of different cultivated members of the Chloroflexus assemblage, whereas nine fell into the T78 group of environmental clones. For the latter subgroup of the green nonsulfur bacteria, no molecular isolate from freshwater plankton has been reported so far. Several of the sequence types occurred in more than one lake, indicating that not only relatives of the Chloroflexus assemblage, but also bacteria of the clone T78 group represent indigenous bacteria of nonthermal stratified freshwater ecosystems. Our results indicate that the natural diversity in the phylum of the green nonsulfur bacteria has been significantly underestimated in the past.
Keywords: Anoxygenic photosynthesis Chloronema Clone T78 group Green nonsulfur bacteria Microbial diversity 16S rRNA
No Title by Giovanna Di Tomaso; Roberto Borghese; Davide Zannoni (pp. 11-19).
This study reports on the construction, calibration and use of recombinant cells of Rhodobacter capsulatus expressing the luciferase gene of the North American firefly Photinus pyralis to detect, by bioluminescence, variations of endogenous ATP levels under various physiological conditions. We show that the antibiotic polymyxin B allows luciferin to rapidly move into cell cytosol, but does not make external ATP freely accessible to intracellular luciferase. Notably, in toluene:ethanol-permeabilized cells, the apparent K mATP for luciferase (50 µM) is similar to that measured in soluble cell fractions. This finding limits the applicability of the firefly luciferase for monitoring intracellular maximal ATP concentration because dark/aerobic-grown recombinant cells of Rba. capsulatus contain approximately 1.3–2.6±0.5 mM ATP. Therefore, the effects of chemical and physical factors such as oxygen, light, carbonyl cyanide m-chlorophenyl hydrazone and antimycin A on ATP synthesis were examined in cells subjected to different starvation periods to reduce the endogenous ATP pool below the luciferase ATP saturation level (≤0.2 mM). We conclude that the amount of endogenous ATP generated by light is maximal in the presence of oxygen, which is required to optimize the membrane redox poise.
Keywords: ATP synthesis Luciferin Polymyxin B Recombinant firefly luciferase Rhodobacter capsulatus
No Title by Rainer Kalscheuer; Marc Wältermann; Hector Alvarez; Alexander Steinbüchel (pp. 20-28).
Triacylglycerol granules synthesized and accumulated by Rhodococcus opacus and Rhodococcus ruber were isolated by glycerol density gradient centrifugation. Whereas only one type of granule could be isolated from R. opacus, two types of granules with different specific densities were isolated from R. ruber. Both types of R. ruber granules showed a similar content of triacylglycerols and poly(3-hydroxybutyrate-co-3-hydroxyvalerate), but the protein profiles of both types were significantly different. The granules with the lower specific density were colorless; the granules with the higher specific density had a deep orange pigmentation. Solubilization studies revealed three different groups of granule-associated proteins: (1) unspecifically bound proteins, (2) relatively weakly associated proteins, and (3) proteins that resisted solubilization by treatment with 2 M NaCl, 2% (w/v) Triton X-114, 6 M guanidinium hydrochloride, up to 8% (w/v) SDS, and proteolytic digestion. The strong association of proteins of the last group suggested that these may play a specific role in the synthesis or mobilization of storage lipids or in the structure of the granules. The N-terminal amino acid sequences of the most tightly bound proteins were obtained. Proteins of low molecular weight with striking sequence similarity to the ribosomal protein L7 from various actinomycetes were always copurified with the granules.
Keywords: Rhodococcus ruber Rhodococcus opacus PHA Polyhydroxyalkanoic acids Triacylglycerols Lipid inclusions Granule-associated proteins
No Title by Gerald Kayingo; Stephanus G. Kilian; Bernard A. Prior (pp. 29-35).
In response to fluctuations in environmental osmolarity, yeast cells adjust their intracellular solute concentrations in order to maintain a constant turgor pressure and ensure continuation of cellular activity. In this study, the effect of hypo-osmotic stress on osmolyte content of osmotolerant yeasts Zygosaccharomyces rouxii and Pichia sorbitophila and the less tolerant Saccharomyes cerevisiae was investigated. All these yeasts released glycerol upon hypo-osmotic shock. However, Z. rouxii also released arabitol, whereas P. sorbitophila released erythritol in addition to arabitol and glycerol. Osmolyte release was very rapid and specific and was neither affected by reduced temperatures nor inhibited by the channel blocker gadolinium or the protonophore carbonyl cyanide m-chlorophenyl hydrazone. Extracellular osmolyte levels increased drastically suggesting that osmolytes were not metabolised but mainly released upon exposure to hypotonic conditions. The export process is well controlled, and the amount of osmolyte released was proportional to the shock intensity. Osmolyte release occurred with little cell lysis and thus the survival as well as the subsequent growth of yeast cells was largely unaffected after hypo-osmotic shock. The kinetics and patterns of osmolyte export suggest the involvement of channel proteins, but the molecular nature of this export pathway in yeasts, with exception of S. cerevisiae, remains to be established.
Keywords: Osmolyte release Conservation Membrane transport Hypo-osmotic stress responses Osmotolerant yeasts
No Title by Osamu Tanaka; Sadahiro Ohmomo (pp. 36-40).
Conditions for protoplast regeneration were examined for several strains of homofermentative lactobacilli and pediococci isolated from silage. Attempts to regenerate protoplasts using previously published agar regeneration media for lactobacilli were unsuccessful for most of the strains. Replacing or increasing colloidal substances in a medium containing raffinose and MgCl2 as osmotic stabilizers enabled efficient regeneration of the protoplasts at a frequency of 10–99%. A medium containing gelatin, polyvinylpyrrolidone (PVP) and no agar was effective for Lactobacillus plantarum, Lactobacillus pentosus and Lactobacillus rhamnosus protoplasts. An agar medium containing PVP (PVP medium) was effective for Pediococcus sp. protoplasts, and addition of agarose to the PVP medium enabled regeneration of Lactobacillus casei protoplasts. A medium containing calcium alginate gel and no agar was effective for Lactobacillus curvatus protoplasts. The type of colloidal substance required for protoplast regeneration varied from species to species. This result suggested that several kinds of media may be necessary to regenerate protoplasts for all the genera of lactobacilli and pediococci.
Keywords: Protoplast regeneration Lactobacillus Pediococcus
No Title by Minna K. Männistö; Jaakko A. Puhakka (pp. 41-46).
Cellular fatty acid compositions of five psychrotolerant groundwater isolates representing α- and β-Proteobacteria were studied at temperatures ranging from 8 to 25 °C. Unsaturation of straight-chain fatty acids was the most common response to decreasing temperature and was detected in four of the isolates. On solid media, decrease of temperature resulted in a decrease of cyclopropane fatty acids in β-proteobacterial isolates. The formation of cyclopropane fatty acids depended, however, to a greater extent on the growth phase than the temperature and increased drastically as the cells entered stationary phase. The α-proteobacterial isolates contained a branched C19:1 fatty acid. The formation of the branched C19:1 increased during growth in the same way as the cyclopropane fatty acids in β-proteobacterial strains, indicating possibly an analogous formation of the branched fatty acid by methylation of the 18:1 fatty acid. Sphingomonas sp. K6 possessed a novel temperature-induced modification of lipid fatty acids. As temperature decreased from 25 to 8 °C, the fatty acid composition shifted from predominantly even-carbon fatty acids to odd-carbon fatty acids. The results show completely different fatty acid modifications in two strains of the same genus Sphingomonas.
Keywords: Fatty acids Groundwater Proteobacteria Psychrotolerant bacteria Temperature adaptation
No Title by Kathleen H. Dannelly; Yanwen Liu; Swapan K. Ghosh (pp. 47-53).
Since the increased use of extended-wear contact lenses, Pseudomonas aeruginosa has emerged as a primary etiological agent of ulcerative keratitis. Clinical isolates have been classified into two types: cytotoxic and non-cytotoxic. This study revealed significant immune and neuro-enzymatic changes elicited by the two types of P. aeruginosa in the lacrimal gland of rats. The humoral immune response in the lacrimal gland to the non-cytotoxic strain was significantly lower than to the cytotoxic strain, possibly due to the immunogenicity of the extracellular toxin; however, the same effect was not seen in the serum. Choline acetyltransferase and acetylcholinesterase are known to be responsible for synthesis and degradation of acetylcholine, respectively, binding receptors on acini and plasma cells, modulating their activity, and constituting the principle regulator of tear secretion. Following infection, neuro-enzyme activities were significantly modified to reduce the concentration of acetylcholine and therefore potentially reduce secretion from the glands. The data lead to the hypothesis that P. aeruginosa may have the potential to reduce the protective barrier provided by the lacrimal gland to benefit pathogenicity. It was also observed that the neuro-enzyme response of the lacrimal glands of uninfected eyes of the test animals was the same as that of infected eyes, implying that the signal may be relayed by common lymphoidal tissue or the central nervous system and a measurable response returned to both eyes.
Keywords: Pseudomonas aeruginosa Lacrimal gland Cholinergic enzymes Neuroimmune interaction
No Title by Natasa Miladinov; Oscar P. Kuipers; Ljubisa Topisirovic (pp. 54-61).
Casitone added to chemically defined medium (CDM) specifically influenced the regulation of the proteinase activity in the natural isolate Lactococcus lactis subsp. lactis BGIS29. Comparative analysis of the influence of casitone present in CDM on the proteolytic activity of strain BGIS29 and of L. lactis subsp. cremoris strains SK11 and Wg2 indicated that the proteolytic activity of strains BGIS29 and SK11 is casitone-dependent, whereas that of strain Wg2 is not. The regulatory region of the prt genes of strain BGIS29 was cloned and sequenced. The nucleotide sequence of the prt regulatory region of strain BGIS29 was distinctly different from that of L. lactis subsp. cremoris strains SK11 and Wg2. Transcriptional gene fusions with the Escherichia coli β-glucuronidase gene (gusA) were used to study medium-dependent expression of two divergent prtP and prtM promoters of strain BGIS29 (designated P prtP and P prtM , respectively). β-Glucuronidase assays and Northern blot analysis showed that the activities of P prtP and P prtM are controlled by casitone at the transcriptional level.
Keywords: Lactococcus lactis Proteinase prtP gene prtM gene Regulation
No Title by Thomas Hansen; Peter Schönheit (pp. 62-69).
The gene (ORF APF0012) encoding the ATP-dependent 6-phosphofructokinase (ATP-PFK) of the hyperthermophilic archaeon Aeropyrum pernix was identified, cloned, and functionally expressed in Escherichia coli. The deduced amino acid sequence showed similarity (25–40%) to members of PFK-B sugar kinases. The purified recombinant enzyme is a heterotetramer of 115 kDa, composed of 34-kDa subunits. Rate dependence (at 85 °C) on both fructose 6-phosphate (F-6-P) and ATP followed Michaelis-Menten kinetics with apparent K m values of 0.25 mM and 0.68 mM, respectively; apparent V max values were about 5 U/mg. The enzyme was specific for ATP as phosphoryl donor, but showed a broader spectrum of phosphoryl acceptors: in addition to F-6-P, glucose 6-phosphate, adenosine, fructose, ribose 5-phosphate, and ribose were accepted. Enzyme activity required divalent cations; Mg2+, which was most effective, could partially be replaced by Co2+, Ni2+, or Mn2+. The enzyme had a temperature optimum of 90 °C and showed a significant thermostability up to 100 °C. ATP-PFK activity was not allosterically regulated by classical effectors of ATP-PFKs of eukarya and bacteria, such as ADP and phosphoenolpyruvate. In accordance, this archaeal ATP-PFK did not contain the typical conserved binding sites for these effectors. This is the first report of a sequence of an archaeal ATP-PFK related to the PFK-B sugar kinase family.
Keywords: Hyperthermophilic archaea Aeropyrum pernix ATP-dependent 6-phosphofructokinase Non-allosteric Sugar kinase PFK-B family PFK-A family
No Title by Eva M. Egelseer; Thomas Danhorn; Magdalena Pleschberger; Christoph Hotzy; Uwe B. Sleytr; Margit Sára (pp. 70-80).
The cell surface of Bacillus stearothermophilus ATCC 12980 is completely covered by an oblique lattice which consists of the S-layer protein SbsC. On SDS-polyacrylamide gels, the mature S-layer protein migrates as a single band with an apparent molecular mass of 122 kDa. During cultivation of B. stearothermophilus ATCC 12980 at 67 °C instead of 55 °C, a variant developed that had a secondary cell wall polymer identical to that of the wild-type strain, but it carried an S-layer glycoprotein that could be separated on SDS-polyacrylamide gels into four bands with apparent molecular masses of 92, 118, 150 and 175 kDa. After deglycosylation, only a single protein band with a molecular mass of 92 kDa remained. The complete nucleotide sequence encoding the protein moiety of this S-layer glycoprotein, termed SbsD, was established by PCR and inverse PCR. The sbsD gene of 2,709 bp is predicted to encode a protein of 96.2 kDa with a 30-amino-acid signal peptide. Within the 807 bp encoding the signal peptide and the N-terminal sequence (amino acids 31–269), different nucleotides for sbsD and sbsC were observed in 46 positions, but 70% of these mutations were silent, thus leading to a level of identity of 95% for the N-terminal parts. The level of identity of the remaining parts of SbsD and SbsC was below 10%, indicating that the lysine-, tyrosine- and arginine-rich N-terminal region in combination with a distinct type of secondary cell wall polymer remained conserved upon S-layer variation. The sbsD sequence encoding the mature S-layer protein cloned into the pET28a vector led to stable expression in Escherichia coli HMS174(DE3). This is the first example demonstrating that S-layer variation leads to the synthesis of an S-layer glycoprotein.
Keywords: S-layer glycoprotein S-layer variation Bacillus stearothermophilus Secondary cell wall polymer Heterologous expression
No Title by Gwénaël Jan; Michel Le Hénaff; Catherine Fontenelle; Henri Wróblewski (pp. 81-90).
Two membrane proteins from the avian pathogen Mycoplasma gallisepticum have been previously purified using a simple, efficient and non-denaturing method: a lipoprotein P67 (pMGA) and P52. In the current study, the lipid part of P67 was chemically analysed. The molecular structure of the lipoprotein-lipid component was determined to be S-glyceryl cysteine with two O-ester-linked acyl chains. Fatty acid analysis of the purified P67 indicated a heterogeneous composition: palmitic acid (16:0)>stearic acid (18:0)>oleic acid (18:1c)>myristic acid (14:0), with 16:0 as the major component. These findings, along with previously published results, support the conclusion that P67 is pMGA1.2, a true membrane-associated lipoprotein although not N-acylated. In contrast to P67, P52 is not a lipoprotein. Topological experiments using in situ treatment with proteases and growth inhibition in the presence of anti-P52 serum provided evidence of the surface exposition of the polypeptide. The N-terminal sequence of P52 was found to be similar to the dihydrolipoamide acetyltransferase from several mollicutes; this enzyme is a membrane-associated component of the pyruvate dehydrogenase complex. Immunoblotting techniques revealed that the surface antigens P52 and P67 were specific to the species M. gallisepticum and the closely related species M. imitans. No antigenic difference was revealed within these species with the anti-P52 serum, while anti-P67 serum confirmed the antigenic variability of P67. The potential of P52 and P67 as antigens in serological diagnosis tests or as candidates for anti-mycoplasma subunit vaccines is discussed.
Keywords: Mollicutes Mycoplasma gallisepticum Surface antigens Lipoproteins ISCOMs
No Title by Sirin A. Adham; Pilar Honrubia; Margarita Díaz; José M. Fernández-Abalos; Ramón I. Santamaría; José A. Gil (pp. 91-97).
The xylanase (xysA) and the cellulase (celA1) genes from Streptomyces halstedii JM8 were cloned into Escherichia coli/Brevibacterium lactofermentum shuttle vectors and successfully expressed in both hosts when placed downstream from the kanamycin resistance promoter (Pkan) from Tn5 but not when under the control of their own promoters. Xylanase was secreted into the culture media of B. lactofermentum by removal of the same leader peptide as is removed in S. halstedii. The main difference between the production of xylanase by Streptomyces and corynebacteria was the low level of processing of the mature extracellular xylanase by B. lactofermentum, probably due to the lack of protease activity in this microorganism.
Keywords: Secretion Corynebacteria Xylanase Cellulase Proteolytic processing Leader peptide
No Title by Beatrica Sevciková; Oldrich Benada; Olga Kofronova; Jan Kormanec (pp. 98-106).
We previously cloned the sigH gene encoding a stress-response sigma factor σΗ in Streptomyces coelicolor A3(2), located in an operon with the gene encoding proposed anti-sigma factor UshX. To clarify the in vivo function of σΗ, a stable null mutant of sigH was prepared by homologous recombination. This mutation appeared to have no obvious effect on vegetative growth, but dramatically affected morphological differentiation. Microscopy showed that the sigH mutant produced undifferentiated hyphae with rare spore chains, giving the colony a pale gray color compared to the dark gray wild-type spores. The sigH mutation partially affected growth under conditions of high osmolarity. Expression of the sigH operon was investigated in the S. coelicolor sigH mutant. Out of four promoters directing expression of the sigH operon, the sigH-P2 promoter – the only promoter preferentially induced by salt-stress conditions – was inactive in the sigH mutant. The results indicated that the sigH-P2 promoter is dependent (directly or indirectly) upon σΗ and that the operon is autocatalytically activated. We propose that in S. coelicolor σΗ has a dual role, regulating the osmotic response and morphological differentiation.
Keywords: Streptomyces Stress response RNA polymerase Sigma factor Anti-sigma-factor Gene disruption Osmotolerance Differentiation
No Title by Grant Buchanan; Frank Sargent; Ben C. Berks; Tracy Palmer (pp. 107-112).
The Escherichia coli Tat protein export pathway transports folded proteins synthesized with N-terminal twin-arginine signal peptides. Twin-arginine signal sequences contain a conserved SRRxFLK "twin-arginine" amino acid sequence motif which is required for protein export by the Tat pathway. The E. coli trimethylamine N-oxide reductase (TorA) is a Tat-dependent periplasmic molybdoenzyme that facilitates anaerobic respiration with trimethylamine N-oxide as terminal electron acceptor. Here, we describe mutant strains constructed with modified TorA twin-arginine signal peptides. Substitution of the second arginine residue of the TorA signal peptide twin-arginine motif with either lysine or aspartate, or the simultaneous substitution of both arginines with lysine residues, completely abolished export. In each case, the now cytoplasmically localised TorA retained full enzymatic activity with the artificial electron donor benzyl viologen. However, the mutant strains were incapable of anaerobic growth with trimethylamine N-oxide and the non-fermentable carbon-source glycerol. The growth phenotype of the mutant strains was exploited in a genetic screen with the aim of identifying second-site suppressor mutations that allowed export of the modified TorA precursors.
Keywords: Escherichia coli Tat protein export pathway Twin-arginine signal peptide TMAO reductase Suppression mutagenesis
No Title by Michael Rother; August Böck; Chris Wyss (pp. 113-116).
Assessment of the nutritional requirements of Treponema denticola disclosed a strict growth dependence on selenium. In vivo labeling of cells of this organism with 75Se and electrophoretic analysis revealed three labeled bands, two of which were selenoproteins correlating in size with subunits A and B of glycine reductase. Antibodies directed against glycine- or betaine-reductase subunits of Eubacterium acidaminophilum specifically also reacted with proteins from cell lysates of T. denticola. Moreover, ORFs within the T. denticola genome sequence were found whose products display high sequence similarity to glycine-reductase subunits. These findings strongly support the notion that T. denticola ferments amino acids via the activity of glycine reductase, an enzyme previously thought to be restricted to gram-positive bacteria.
Keywords: Treponema denticola Selenium Glycine reductase
No Title by Svetlana N. Dedysh; Hans-Peter Horz; Peter F. Dunfield; Werner Liesack (pp. 117-121).
A fragment of the functional gene pmoA, which encodes the active-site polypeptide of particulate methane monooxygenase (pMMO), was PCR-amplified from DNA of the recently described acidophilic methanotrophic bacterium Methylocapsa acidophila B2. This methanotroph was isolated from an acidic Sphagnum peat bog and possesses a novel type III arrangement of intracytoplasmic membranes. Comparative sequence analysis revealed that the inferred peptide sequence of pmoA of Methylocapsa acidophila B2 belongs to a novel PmoA lineage. This lineage was only distantly related to the PmoA sequence cluster of type II methanotrophs, with identity values between 69.5% and 72%. The identity values between the PmoA of Methylocapsa acidophila B2 and PmoA sequences of type I methanotrophs ranged from 55.5 to 68%. However, the PmoA of this acidophilic methanotroph was more closely affiliated with the inferred peptide sequences of pmoA clones retrieved from various acidic upland soils showing atmospheric methane consumption. The PmoA identity values with these clones were 79.5–81%. Nonetheless, the apparent affinity for methane exhibited by Methylocapsa acidophila B2 was 1–2 µM, which is similar to values measured in other methanotrophic bacteria. This finding suggests that the pMMO of Methylocapsa acidophila B2 is not a novel enzyme specialized to have a high affinity for methane and that apparent "high-affinity" methane uptake is either the result of particular culture conditions or is performed by another pMMO type.
Keywords: pmoA Methylocapsa acidophila B2 Methanotrophic bacteria Methane affinity