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Archives of Microbiology (v.174, #1-2)
No Title by Mark A. Smith; Moshe Finel; Victoria Korolik; George L. Mendz (pp. 1-10).
The respiratory chain enzymes of microaerophilic bacteria should play a major role in their adaptation to growth at low oxygen tensions. The genes encoding the putative NADH:quinone reductases (NDH-1), the ubiquinol:cytochrome c oxidoreductases (bc 1 complex) and the terminal oxidases of the microaerophiles Campylobacter jejuni and Helicobacter pylori were analysed to identify structural elements that may be required for their unique energy metabolism. The gene clusters encoding NDH-1 in both C. jejuni and H. pylori lacked nuoE and nuoF, and in their place were genes encoding two unknown proteins. The NuoG subunit in these microaerophilic bacteria appeared to have an additional Fe-S cluster that is not present in NDH-1 from other organisms; but C. jejuni and H. pylori differed from each other in a cysteine-rich segment in this subunit, which is present in some but not all NDH-1. Both organisms lacked genes orthologous to those encoding NDH-2. The subunits of the bc 1 complex of both bacteria were similar, and the Rieske Fe-S and cytochrome b subunits had significant similarity to those of Paracoccus denitrificans and Rhodobacter capsulatus, well-studied bacterial bc 1 complexes. The composition of the terminal oxidases of C. jejuni and H. pylori was different; both bacteria had cytochrome cbb 3 oxidases, but C. jejuni also contained a bd-type quinol oxidase. The primary structures of the major subunits of the cbb 3-type (terminal) oxidase of C. jejuni and H. pylori indicated that they form a separate group within the cbb 3 protein family. The implications of the results for the function of the enzymes and their adaptation to microaerophilic growth are discussed.
Keywords: Respiratory chain Microaerophiles Campylobacter jejuni Helicobacter pylori
No Title by E. Hoiczyk (pp. 11-17).
Cyanobacteria are a morphologically diverse group of phototrophic prokaryotes that are capable of a peculiar type of motility characterized as gliding. Gliding motility requires contact with a solid surface and occurs in a direction parallel to the long axis of the cell or filament. Although the mechanistic basis for gliding motility in cyanobacteria has not been established, recent ultrastructural work has helped to identify characteristic structural features that may play a role in this type of locomotion. Among these features are the distinct cell surfaces formed by specifically arranged protein fibrils and organelle-like structures, which may be involved in the secretion of mucilage during locomotion. The possible role of these ultrastructural features, as well as consequences for understanding the molecular basis of gliding motility in cyanobacteria, are the topic of this review.
Keywords: Cyanobacteria Gliding motility Bacterial surface proteins Carbohydrate secretion Bacterial organelles
No Title by Amy D. Milford; Laurie A. Achenbach; Deborah O. Jung; Michael T. Madigan (pp. 18-27).
From enrichment cultures established for purple nonsulfur bacteria using water and sediment samples from Lake Bogoria and Crater Lake, two soda lakes in the African Rift Valley, three strains of purple nonsulfur bacteria were isolated; strain LBB1 was studied in detail. Cells of strain LBB1 were motile and spherical to rod-shaped, suggesting a relationship to Rhodobacter or Rhodovulum species, and the organism was capable of both phototrophic and chemotrophic growth on a wide variety of organic compounds. Phototrophically grown cultures were yellow to yellow-brown in color and grew optimally at pH 9 (pH range 7.5–10) and 1% NaCl (range 0–10%). In physiological studies of strain LBB1, neither photoautotrophy (H2- or sulfide-dependent) nor nitrogen fixation was observed. Absorption spectra revealed that all three strains contained bacteriochlorophyll a and carotenoids of the spheroidene pathway and synthesized only a light-harvesting (LH) I-type photosynthetic antenna complex. Electron microscopy of cells of strain LBB1 revealed a vesicular intracytoplasmic membrane system, although only a few vesicles were observed per cell. The G+C content of strain LBB1 DNA was 59 mol%, significantly lower than that of known Rhodobacter and Rhodovulum species, and its phylogeny as determined by ribosomal RNA gene sequencing placed it within the Rhodobacter/Rhodovulum clade yet distinct from all described species of either of these genera. The unique assemblage of properties observed in strain LBB1 warrants its inclusion in a new genus of purple nonsulfur bacteria and the name Rhodobaca bogoriensis is proposed herein, the genus name reflecting morphological characteristics and the species epithet referring to the habitat.
Keywords: Photosynthesis Anoxygenic phototrophic bacteria Purple nonsulfur bacteria Extreme environments Alkaliphily
No Title by June-Nam Seah; Jimmy Kwang (pp. 28-34).
The H serogroup of Escherichia coli is determined by the flagellar antigen, flagellin. Sequence analysis of the flagellin gene, fliC, reveals a central variable region and the highly conserved N- and C-termini. This variable region has been shown to encode both H-specific and cross-reactive epitopes. Using polyclonal antibodies, we mapped the linear H-specific determinants in flagellin from four E. coli serotypes O157:H10, O138:H14, O157:H42 and O157:H43. The specificity of all potential fragments was verified with 52 ECRC (Escherichia coli Reference Center) H-specific antisera. Our results indicated that: (a) a specific determinant of H10 flagellin (1263 bp long) maps to the region covering amino acid residues 305–331; (b) a specific determinant of H14 flagellin (1653 bp long) maps to the region covering amino acid residues 430–461; (c) a specific determinant of H42 flagellin (1281 bp long) maps to a region covering amino acid residues 171–201; and (d) a specific determinant of H43 flagellin (1506 bp long) maps to a region covering amino acid residues 200–260.
Keywords: Escherichia coli Flagella Flagellin fliC Mapping Serotype
No Title by Angela S. Lindner; Peter Adriaens; Jeremy D. Semrau (pp. 35-41).
The ability of Methylosinus trichosporium OB3b, expressing soluble methane monooxygenase, to oxidize a range of ortho-substituted biphenyls was examined to better understand how substituents affect both the rate and products of oxidation in comparison to biphenyl. Inhibition of oxidation was observed over the tested substrate range for both biphenyl and ortho-halogenated biphenyls (2-chloro-, 2-bromo-, and 2-iodobiphenyl). No inhibition was observed during the oxidation of 2-hydroxybiphenyl and 2-methylbiphenyl. Analysis of the products of oxidation showed that, depending on the substituent, ring hydroxylation, substituent oxidation, and elimination pathways could occur. The type and abundance of products formed along with the relatively high kinetic isotope effect observed for deuterated vs. nondeuterated biphenyl (k h/k d = 3.4±0.02) are consistent with mechanisms that include both hydrogen abstraction and NIH-shift pathways. Knowledge of these substituent-dependent reaction rates and mechanisms enhances our understanding of the methanotrophic aryl transformation potential and allows for better prediction of the formation of oxidized intermediates by methanotrophic bacteria.
Keywords: Methanotrophs Soluble methane monooxygenase Substituted biphenyls Biodegradation
No Title by Silvana Povolo; Sergio Casella (pp. 42-49).
During free-living reproductive growth, Sinorhizobium meliloti accumulates poly-β-hydroxybutyrate (PHB) and glycogen, and produces and excretes exopolysaccharides and β-1,2-glucan. In previous investigations, PHB-minus mutants of S. meliloti 41 were obtained and studied; and the genes for PHB biosynthesis, phaAB and phaC, were described. In this work, the role of an open reading frame (orf) upstream of phaAB is studied. This orf is designated aniA because the gene was found to be expressed during anaerobic growth. Under low oxygen conditions, glycogen decreases and the production of extracellular polymeric substances (EPS) is partially repressed. When the aniA mutant is incubated under oxygen-limiting conditions, the only significant change observed is an overproduction of EPS. Subsequent in planta tests showed that although the mutant strain produced abundant nodules, only very low acetylene-reduction activity was detected, indicating that nitrogen fixation was not adequately supported by endogenous substrates.
Keywords: Sinorhizobium meliloti Poly-β-hydroxybutyrate Extracellular polymeric substances Nitrogen fixation aniA
No Title by Jürgen M. Fröstl; Jörg Overmann (pp. 50-58).
The phylogenetic affiliation of epibionts occurring in three morphologically distinct types of green-colored phototrophic consortia was investigated. Intact consortia of the types "Chlorochromatium aggregatum", "C. glebulum", and a third previously undescribed type, tentatively named "C. magnum" were mechanically separated from accompanying bacteria by either micromanipulation or by chemotactic accumulation in sulfide-containing capillaries. A 540-base-pair-long fragment of the 16S rRNA gene of the epibionts was amplified employing PCR primers specific for green sulfur bacteria. DNA fragments were separated by denaturing gradient gel electrophoresis and subsequently sequenced. The results of this phylogenetic analysis indicated that the symbiotic epibionts, together with only a few free-living strains, form a cluster within the green sulfur bacterial radiation which is only distantly related to the majority of known representatives of this phylum. Consortia with identical morphology but different origin exhibited significant differences in their partial 16S rRNA gene sequences, which could be confirmed by analysis of the 16S rRNA secondary structure. The phylogenetic affiliation of the chemotrophic central rod-shaped bacterium of "C. aggregatum" and "C. magnum" was analyzed by fluorescent in situ hybridization. According to our results and contrary to earlier assumptions, the central bacterium is a member of the β-subgroup of the Proteobacteria.
Keywords: Phototrophic consortia "Chlorochromatium" Green Sulfur bacteria β-Proteobacteria Micromanipulation Chemotaxis
No Title by Cristina Amor; Ana I. Domínguez; J. Ramón De Lucas; Fernando Laborda (pp. 59-66).
In Aspergillus nidulans, activity of the glyoxylate cycle enzyme isocitrate lyase is finely regulated. Isocitrate lyase is induced by growth on C2 compounds and long-chain fatty acids and repressed by glucose. In addition, activity of isocitrate lyase is subject to a second mechanism of catabolite control, glucose-induced inactivation. Here, we demonstrate that the catabolite inactivation of A. nidulans isocitrate lyase, a process that takes place during glucose adaptation of cells grown under gluconeogenic conditions, occurs by proteolysis of the enzyme. Ultrastructural analyses were carried out in order to investigate the cellular processes that govern the catabolite inactivation of this peroxisomal enzyme. Addition of glucose to oleate-induced cells triggered the specific engulfment and sequestration of peroxisomes by the vacuoles. Sequestration of various peroxisomes by a single vacuole was a frequently observed phenomenon. Results obtained by immunoelectron microscopy using antibodies against A. nidulans isocitrate lyase showed that degradation of this peroxisomal enzyme occurred inside the vacuole. In addition, ultrastructural studies demonstrated that microautophagy was the autophagic pathway involved in degradation of redundant peroxisomes during glucose adaptation of oleate-induced cells of A. nidulans.
Keywords: Aspergillus nidulans Isocitrate lyase Glyoxylate cycle Glucose inactivation Vacuoles Peroxisomes Microbodies Autophagy Microautophagy
No Title by Christopher N. Kästner; Peter Dimroth; Klaas M. Pos (pp. 67-73).
The Na+-dependent citrate carrier of Klebsiella pneumoniae (CitS) is a member of the 2-hydroxycarboxylate transporter family. Within the highly conserved helix Vb region, Asn-185 of CitS was mutated to Val and Glu-194 was mutated to Gln. The wild-type and mutant proteins were synthesised in Escherichia coli DH5α or C43(DE3) as biotinylated or His-tagged CitS-fusions, respectively. The synthesis and purification procedure yielded 6.5 mg pure CitS per litre culture. The fusion proteins were characterised with E. coli cell suspensions or after reconstitution of the purified enzymes into proteoliposomes. The E194Q mutation had almost no effect on the kinetics of Na+ or citrate transport. In contrast, aberrant citrate transport kinetics were found for the N185V mutant. The apparent K m value for the citrate species H-citrate2- was increased about nine-fold, whereas the apparent V max value and the effect of Na+ on the transport kinetics were comparable to the wild-type. Asn-185 of CitS appears therefore to participate in the binding of H-citrate2–.
Keywords: citS gene CitS citrate carrier Secondary active transporter Sodium ion/citrate symporter Mutagenesis
No Title by Maddalena Rossi; Luigi Altomare; Antonio Gonzàlez Vara y Rodriguez; Patrizia Brigidi; Diego Matteuzzi (pp. 74-80).
The gene encoding β-galactosidase was isolated by functional complementation of Escherichia coli from Bifidobacterium longum MB219, which exhibited the highest activity among ten Bifidobacterium strains tested of the species B. longum, B. breve, B. adolescentis, B. indicum, B. animalis and B. cuniculi. The nucleotide sequence of the 5.0-kb fragment conferring the positive β-galactosidase phenotype to E. coli revealed the presence of a lacZ-type gene encoding a 1023-amino-acid protein that was preceded by a ribosome binding site. A sequence showing 72% identity with the proline tRNA of Bacillus subtilis and a gene probably encoding the DNA-3-methyladenine glycosydase I were located downstream from the lacZ gene, after a gap of 30–50 unsequenced base pairs. By primer-extension analysis, the transcription start site of the lacZ gene was mapped 65 nt upstream from the start codon, and it enabled identification of the –10 region of the putative promoter. The nucleotide sequence of lacZ and its deduced amino acid sequence were compared with those of β-galactosidase genes and enzymes from other microorganisms. High similarity was demonstrated between the B. longum β-galactosidase and its counterparts in Lactobacillus delbruckii subsp. bulgaricus, Streptococcus salivarius subsp. thermophilus, E. coli, Clostridium acetobutylicum, Leuconostoc lactis, Klebsiella pneumoniae and Kluyveromyces marxianus var. lactis, all belonging to the LacZ family. The B. longum MB219 lacZ gene was cloned in Bifidobacterium and its expression was observed in strains with otherwise low levels of endogenous activity. The expression increased by factors of 1.5–50 and enabled those strains that do not grow on lactose to use this sugar as sole carbon source.
Keywords: Bifidobacterium longum LacZ Expression Transcriptional analysis Nucleotide sequence
No Title by Takamasa Tobimatsu; Hideki Kajiura; Tetsuo Toraya (pp. 81-88).
Adenosylcobalamin-dependent glycerol and diol dehydratases undergo inactivation by the physiological substrate glycerol during catalysis. In the permeabilized cells of Klebsiella pneumoniae, Klebsiella oxytoca, and recombinant Escherichia coli, glycerol-inactivated glycerol dehydratase and diol dehydratase are reactivated by their respective reactivating factors in the presence of ATP, Mg2+, and adenosylcobalamin. Both of the reactivating factors consist of two subunits. To examine the specificities of the reactivating factors, their genes or their hybrid genes were co-expressed with dehydratase genes in E. coli cells in various combinations. The reactivating factor of K. oxytoca for diol dehydratase efficiently cross-reactivated the inactivated glycerol dehydratase, whereas the reactivating factor of K. pneumoniae for glycerol dehydratase hardly cross-reactivated the inactivated diol dehydratase. Both of the two hybrid reactivating factors rapidly reactivated the inactivated glycerol dehydratase. In contrast, the hybrid reactivating factor containing the large subunit of the glycerol dehydratase reactivating factor hardly reactivated the inactivated diol dehydratase. These results indicate that the glycerol dehydratase reactivating factor is much more specific for the dehydratase partner than the diol dehydratase reactivating factor and that a large subunit of the reactivating factors principally determines the specificity for a dehydratase.
Keywords: Adenosylcobalamin Coenzyme B12 Glycerol dehydratase Klebsiella pneumoniae Diol dehydratase Klebsiella oxytoca Reactivating factor In situ reactivation Coexpression
No Title by Alicja Węgrzyn; Agata Czyż; Magdalena Gabig; Grzegorz Węgrzyn (pp. 89-96).
The O protein is a replication initiator that binds to the oriλ region and promotes assembly of the bacteriophage λ replication complex. This protein, although protected from proteases by other elements of the replication complex, in a free form is rapidly degraded in the host, Escherichia coli, by the ClpP/ClpX protease. Nevertheless, the physiological role of this rapid degradation remains unclear. Here we demonstrate that the copy number of plasmids derived from bacteriophage λ is significantly higher in wild-type cells growing in rich media than in slowly growing bacteria. However, λ plasmid copy number in bacteria devoid of the ClpP/ClpX protease was not dependent on the bacterial growth rate and in all minimal media tested was comparable to that observed in wild-type cells growing in a rich medium. Contrary to λ plasmid replication, the efficiency of lytic growth of bacteriophage λ was found to be dependent on the host growth rate in both wild-type bacteria and clpP and clpX mutants. The activities of two major λ promoters operating during the lytic development, p R and p L, were found to be slightly dependent on the host growth rate. However, when p R activity was significantly decreased in the dnaA mutant, production of phage progeny was completely abolished at low growth rates. These results indicate that the O protein (whose level in E. coli cells depends on the activity of ClpP/ClpX protease) is a major limiting factor in the regulation of λ plasmid replication at low bacterial growth rates. However, this protein seems to be only one of the limiting factors in the bacteriophage λ lytic development under poor growth conditions of host cells. Therefore, it seems that the role of the rapid ClpP/ClpX-mediated proteolysis of the O protein is to decrease the efficiency of early DNA replication of the phage in slowly growing host cells.
Keywords: Bacteriophage λ development Bacterial growth rate λ O protein ClpP/ClpX protease λ Plasmids DNA replication
No Title by Holger Scholz; Susanne Hummel; Angela Witte; Werner Lubitz; Beatrix Kuen (pp. 97-103).
Cell surface (S)-layer protein synthesis in Bacillus stearothermophilus PV72/p6 is blocked when cells are grown at elevated temperature. From a culture exhibiting the S-layer-negative phenotype, the S-layer deficient mutant T5 (SbsA–) was isolated. Genetic analysis of the S-layer-encoding gene (sbsA) of mutant T5 revealed an insertion element (IS4712) integrated into the upstream regulatory region of the S-layer gene, thereby blocking sbsA transcription. The insertion element consists of 1371 base pairs which are flanked by two perfect inverted terminal repeats. Sequence similarity to other transposases of the IS4 family was detected. DNA-DNA hybridizations demonstrated that multiple homologues of IS4712 were also present within the genomes of several other thermophilic bacillus isolates. Attempts to isolate SbsA+ revertants failed. Instead, cells with altered surface proteins were detected. The synthesis of the altered S-layer proteins was correlated with the presence of IS4712 along with the occurrence of deletions in the sbsA coding region. Furthermore imprecise excision of IS4712 was detected. This work demonstrated that B. stearothermophilus is able to express at least four different S-layer proteins and that blocking of sbsA transcription by the insertion element IS4712 is associated with the expression of altered surface proteins.
Keywords: Bacillus stearothermophilus Insertion element S-layer S-layer variation
No Title by Guadalupe Oliva; Irma Romero; Guadalupe Ayala; Idanelli Barrios-Jacobo; Heliodoro Celis (pp. 104-110).
A cytoplasmic pyrophosphatase [E.C. 3.6.1.1.] was partially purified from Helicobacter pylori. The molecular mass was estimated to be 103 kDa by gel filtration. Results of SDS-PAGE suggested that the enzyme consists of six identical subunits of 19.1 kDa each. The enzyme specifically catalyzed the hydrolysis of pyrophosphate and was very sensitive to NaF, but not to sodium molybdate. The optimal pH for activity was 8.5. Mg2+ was required for maximal activity; Mn2+, Co2+, and Zn2+ poorly supported hydrolytic activity. The pyrophosphatase had an apparent K m for Mg-PPi 2– hydrolysis of 90 µM, and a V max estimated at 24.0 µmol Pi min–1 mg–1.
Keywords: Cytoplasmic inorganic pyrophosphatase Helicobacter pylori Fluoride sensitivity
No Title by E. Anne Greene; Perrin H. Beatty; Phillip M. Fedorak (pp. 111-119).
Sulfolane (tetrahydrothiophene-1,1-dioxide) is used in the Sulfinol process for natural gas sweetening. At many sour-gas processing plants spills, landfills and leakage from unlined surface storage ponds have contaminated groundwaters with sulfolane. Due to its high water solubility and mobility in aquifers, sulfolane poses a risk for off-site contamination. This study investigated the aerobic biodegradation of sulfolane by two mixed microbial enrichment cultures and by three bacterial isolates. Sulfolane served as the sole C, S and energy source for these cultures. In the two mixed cultures, 60% and 80% of the sulfolane C was recovered as CO2, whereas in cultures of the three isolates only 40–42% of the substrate C was recovered as CO2. In the mixed cultures, 81% and 97% of the sulfolane S was converted to sulfate, and in the pure isolates, 55–90% of the substrate S was converted to sulfate. Thus, the mixed cultures were capable of greater mineralization than the pure isolates. One isolate, strain WP1, was identified using a combination of 16S rRNA gene sequencing, physiological traits and cell morphology. WP1 was determined to be most similar to Variovorax paradoxus.
Keywords: Biodegradation Natural gas Sulfolane Tetrahydrothiophene-1,1-dioxide Variovorax
No Title by Lázaro Hernández; Mailin Sotolongo; Yamilka Rosabal; Carmen Menéndez; Ricardo Ramírez; Jesús Caballero-Mellado; Juan Arrieta (pp. 120-124).
Levansucrase (EC 2.4.1.10) was identified as a constitutive exoenzyme in 14 Gluconacetobacter diazotrophicus strains recovered from different host plants in diverse geographical regions. The enzyme, consisting of a single 60-kDa polypeptide, hydrolysed sucrose to synthesise oligofructans and levan. Sugar-cane-associated strains of the most abundant genotype (electrophoretic type 1) showed maximal values of levansucrase production. These values were three-fold higher than those of the isolates recovered from coffee plants. Restriction fragment length polymorphism analysis revealed a high degree of conservation of the levansucrase locus (lsdA) among the 14 strains under study, which represented 11 different G. diazotrophicus genotypes. Targeted disruption of the lsdA gene in four representative strains abolished their ability to grow on sucrose, indicating that the endophytic species G. diazotrophicus utilises plant sucrose via levansucrase.
Keywords: Gluconacetobacter diazotrophicus Acetobacter diazotrophicus Endophytic bacteria Levansucrase LsdA Sucrose metabolism Sugar cane
No Title by Nils Arneborg; Lene Jespersen; Mogens Jakobsen (pp. 125-128).
The effects of perfusion with 2.7 and 26 mM undissociated acetic acid in the absence or presence of glucose on short-term intracellular pH (pHi) changes in individual Saccharomyces cerevisiae and Zygosaccharomyces bailii cells were studied using fluorescence-ratio-imaging microscopy and a perfusion system. In the S. cerevisiae cells, perfusion with acetic acid induced strong short-term pHi responses, which were dependent on the undissociated acetic acid concentration and the presence of glucose in the perfusion solutions. In the Z. bailii cells, perfusion with acetic acid induced only very weak short-term pHi responses, which were neither dependent on the undissociated acetic acid concentration nor on the presence of glucose in the perfusion solutions. These results clearly show that Z. bailii is more resistant than S. cerevisiae to short-term pHi changes caused by acetic acid.
Keywords: Saccharomyces cerevisiae Zygosaccharomyces bailii Fluorescence-ratio-imaging microscopy Short-term pHi response Acetic acid Individual cells
No Title by P. Lindberg; A. Hansel; P. Lindblad (pp. 129-133).
The heterocystous cyanobacterium Nostoc sp. PCC 73102 possesses an uptake hydrogenase encoded by hupS and hupL. The genes are transcribed in cells grown under N2-fixing but not under non-N2-fixing conditions. We characterised the transcription of hupS and hupL in further detail. A transcription start site was located at a position 259 bp upstream from the hupS translation start. As shown by RT-PCR, hupS and hupL constitute a single transcript in Nostoc sp. PCC 73102. The possible function of short, tandemly repeated repetitive sequences between hupS and hupL, enabling a folding of the entire intergenic region into a hairpin, is discussed.
Keywords: Cyanobacteria Nostoc Uptake hydrogenase hupSL Transcription 5′-RACE RT-PCR STRR