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Archives of Microbiology (v.179, #6)

Spontaneous chromosome circularization and amplification of a new amplifiable unit of DNA belonging to the terminal inverted repeats in Streptomyces ambofaciens ATCC 23877 by Sibel Catakli; Axelle Andrieux; Pierre Leblond; Bernard Decaris; Annie Dary (pp. 387-393).

In Streptomyces, the linear chromosomal DNA is highly unstable and undergoes large rearrangements usually at the extremities. These rearrangements consist of the deletion of several hundred kilobases, often associated with the amplification of an adjacent sequence, AUD (amplifiable unit of DNA). In Streptomyces ambofaciens, two amplifiable regions (AUD6 and AUD90), located approximately 600 kb and 1,200 kb from the right chromosomal end respectively, have been characterized. Here, the isolation and molecular characterization of a new S. ambofaciens mutant strain exhibiting a green-pigmented phenotype is described; the wild-type produces a gray pigment. In this mutant, both chromosome ends were deleted, which probably led to circularization of the chromosome. These deletions were associated with amplification of a sequence belonging to the chromosomal terminal inverted repeats (TIRs), which might constitute the new fragment generated by the chromosomal circularization.

Keywords: Streptomyces Genetic instability Chromosome circularization DNA amplification

Palaeococcus helgesonii sp. nov., a facultatively anaerobic, hyperthermophilic archaeon from a geothermal well on Vulcano Island, Italy by Jan P. Amend; D'Arcy R. Meyer-Dombard; Seema N. Sheth; Natalya Zolotova; Andrea C. Amend (pp. 394-401).

A novel, hyperthermophilic archaeon was isolated from a shallow geothermal well that taps marine waters on the Island of Vulcano in the southern Tyrrhenian Sea, Italy. The cells were irregular cocci, 0.6–1.5 μm in diameter, with multiple polar flagella. Growth was observed at temperatures from 45 to 85 °C (optimum at ~80 °C), pH 5–8 (optimum at 6.5), and 0.5–6.0% NaCl (optimum at ~2.8%). The minimum doubling time was 50 min. The isolate was obligately chemoheterotrophic, utilizing complex organic compounds including yeast or beef extract, peptone, tryptone, or casein for best growth. The presence of elemental sulfur enhanced growth. The isolate grew anaerobically as well as microaerobically. The G+C content of the genomic DNA was 42.5 mol%. The 16S rRNA sequence indicated that the new isolate was a member of the Thermococcales within the euryarchaeota, representing the second species in the genus Palaeococcus. Its physiology and phylogeny differed in several key characteristics from those of Palaeococcus ferrophilus, justifying the establishment of a new species; the name Palaeococcus helgesonii sp. nov. is proposed, type strain PI1 (DSM 15127).

Keywords: Palaeococcus Hyperthermophilic archaeon Shallow marine hydrothermal system Facultative anaerobe Microaerophilic growth

Purification and characterisation of a lactococcal aminoacylase by Peter Curley; Chris van der Does; Arnold J. M. Driessen; Jan Kok; D. van Sinderen (pp. 402-408).

The amd1-encoded aminoacylase from Lactococcus lactis MG1363 was cloned and overexpressed in Escherichia coli and purified. The assumed dimeric enzyme has a subunit molecular mass of about 42 kDa and contains 2.0±0.1 g-atoms of zinc and cobalt, in equimolar amounts, per subunit of Amd1. The enzyme was characterised with respect to substrate specificity, pH, temperature and metal dependence. Amd1 exhibited a broad activity range towards N-acetylated-l-amino acids with a strong preference towards those containing neutral aliphatic and aromatic side chains. It hydrolysed N-acetyl-l-alanine most efficiently, and exhibited temperature and pH optima of 30 °C and 7.0, respectively. The activity of Amd1 towards N-acetyl-l-alanine was enhanced by the divalent cation Co²⁺, while Cd²⁺inhibited activity. Interestingly, Amd1 was shown to catalyse the hydrolysis of several dipeptides at pH 7.0, although with reduced V _max values as compared to hydrolysis of N-acetylated-l-amino acids. This characteristic has also biological significance since Amd1 was able to complement a growth deficiency in a L. lactis triple peptidase mutant.

Keywords: Lactococcus lactis Aminoacylase Purification Kinetic analysis

A novel type of lycopene ε-cyclase in the marine cyanobacterium Prochlorococcus marinus MED4 by Per Stickforth; Sabine Steiger; Wolfgang R. Hess; Gerhard Sandmann (pp. 409-415).

Chlorophyll-b-possessing cyanobacteria of the genus Prochlorococcus share the presence of high amounts of α- and β-carotenoids with green algae and higher plants. The branch point in carotenoid biosynthesis is the cyclization of lycopene, for which in higher plants two distinct enzymes are required, ε- and β-lycopene cyclase. All cyanobacteria studied so far possess a single β-cyclase. Here, two different Prochlorococcus sp. MED4 genes were functionally identified by heterologous gene complementation in Escherichia coli to encode lycopene cyclases. Whereas one is both functionally and in sequence highly similar to the β-cyclase of Synechococcus sp. strain PCC 7942 and other cyanobacteria, the other showed several intriguing features. It acts as a bifunctional enzyme catalyzing the formation of ε- as well as of β-ionone end groups. Expression of this cyclase in E. coli resulted in the simultaneous accumulation of α- β-, δ-, and ε-carotene. Such an activity is in contrast to all lycopene ε-cyclases known so far, including those of the higher plants. Thus, for the first time among prokaryotes, two individual enzymes were identified in one organism that are responsible for the formation of cyclic carotenoids with either β- or ε-end groups. These two genes are suggested to be designated as crtL-b and crtL-e. The results indicate that both enzymes might have originated from duplication of a single gene. Consequently, we suggest that multiple gene duplications followed by functional diversification resulted several times, and in independent lineages, in the appearance of enzymes for the biosynthesis of cyclic carotenoids.

Keywords: α-Carotene ε-Carotene Lycopene β-cyclase Lycopene ε-cyclase Phytoplankton Protein-pigment complexes

Functional analysis of the calcineurin-encoding gene cnaA from Aspergillus oryzae: evidence for its putative role in stress adaptation by Praveen Rao Juvvadi; Yutaka Kuroki; Manabu Arioka; Harushi Nakajima; Katsuhiko Kitamoto (pp. 416-422).

The presence of putative STRE (stress response regulatory element) and HSF (heat-shock factor) transcription factor binding sites in the promoter region of the gene encoding calcineurin (cnaA) from Aspergillus oryzae implicated a probable role for calcineurin in the stress response. The activity of calcineurin was enhanced during growth of the wild-type strain in the presence of 1 M NaCl (2.6-fold), at alkaline pH 10.0 (2.9-fold) and at 37 °C (1.6-fold). The induction of cnaA antisense expression resulted in reduced calcineurin activity (1.4-fold) and caused a growth defect under the stress conditions. Induction of a strain overexpressing cnaA resulted in an increase in calcineurin activity under stress conditions, such as the presence of 1 M NaCl (73%), alkaline pH 10.0 (70%), and a temperature of 37 °C (50%), in addition to tolerance to FK506 (a specific inhibitor of calcineurin). While a role for calcineurin in hyphal growth is well recognized, the present study suggests that stress adaptation mechanisms in filamentous fungi involve calmodulin/calcineurin-mediated signal transduction pathways.

Keywords: Aspergillus oryzae Stress Calcineurin A cnaA Protein phosphatase 2B Calmodulin- dependent protein phosphatase

Heat-shock response and its contribution to thermotolerance of the nitrogen-fixing cyanobacterium Anabaena sp. strain L-31 by Hema Rajaram; Shree Kumar Apte (pp. 423-429).

Compared to Escherichia coli, the nitrogen-fixing soil cyanobacterium Anabaena sp. strain L-31 exhibited significantly superior abilities to survive prolonged and continuous heat stress and recover therefrom. Temperature upshift induced the synthesis of heat-shock proteins of similar molecular mass in the two microbes. However, in Anabaena sp. strain L-31 the heat-shock proteins (particularly the GroEL proteins) were synthesised throughout the stress period, were much more stable and accumulated during heat stress. In contrast, in E. coli the heat-shock proteins were transiently synthesised, quickly turned over and did not accumulate. Nitrogenase activity of Anabaena cells of sp. strain L-31 continuously exposed to heat stress for 7 days rapidly recovered from thermal injury, although growth recovery was delayed. Exposure of E. coli cells to >4.5 h of heat stress resulted in a complete loss of viability and the ability to recover. Marked differences in the synthesis, stability and accumulation of heat-shock proteins appear to distinguish these bacteria in their thermotolerance and recovery from heat stress.

Keywords: Heat shock response Thermotolerance Anabaena Escherichia coli GroEL

Cleavage of CD14 and LBP by a protease from Prevotella intermedia by James Deschner; Anuradha Singhal; Ping Long; Chau-Ching Liu; Nicholas Piesco; Sudha Agarwal (pp. 430-436).

Periodontitis is an inflammatory disease caused by subgingival microorganisms and their components, such as lipopolysaccharide (LPS). Responses of the host to LPS are mediated by CD14 and LPS-binding protein (LBP). In this study, it was determined that proteases from a periodontal pathogen, Prevotella intermedia, cleave CD14 and LBP, and thereby modulate the virulence of LPS. Culture supernatants from two strains of P. intermedia (ATCC 25611 and 25261) cleaved CD14 and LBP in a concentration-dependent manner. Zymographic and molecular mass analysis revealed the presence of a membrane-associated, 170-kDa, monomeric protease. Class-specific inhibitors and stimulators demonstrated that this enzyme is a metal-requiring, thiol-activated, cysteine protease. The protease was stable over a wide range of temperatures (4–56 °C) and pH values (4.5–8.5). This enzyme also decreased the expression of interleukin-1β (IL-1β)-specific mRNA in the LPS-activated macrophage-like cell lines U937 and THP-1 in a concentration-dependent manner, indicating that it also cleaves membrane-associated CD14. Furthermore, addition of soluble CD14 abrogated protease-mediated inhibition of IL-1 mRNA expression induced by LPS. The observations suggest that proteolysis of CD14 and LBP by P. intermedia protease might modulate the virulence of LPS at sites of periodontal infections.

Keywords: Prevotella intermedia Cysteine protease Lipopolysaccharide Lipopolysaccharide-binding protein CD14 Periodontitis

Encystment and alkylresorcinol production by Azotobacter vinelandii strains impaired in poly-β-hydroxybutyrate synthesis by Daniel Segura; Tania Cruz; Guadalupe Espín (pp. 437-443).

The lipids poly-β-hydroxybutyrate (PHB) and alkylresorcinols are the major metabolic products of Azotobacter vinelandii cysts. Cysts are formed in less than 0.01% of late stationary phase cells grown on sucrose. Culturing vegetative cells in n-butanol or β-hydroxybutyrate induces encystment. After induction of encystment, PHB rapidly accumulates in large granules. Then, the cells begin the synthesis of alkylresorcinols that replace the phospholipids in the membranes and are components of the exine, the outer layer of the cyst envelope. Vegetative cells do not synthesize alkylresorcinols. We report here the effect of mutations in the phbBAC operon, coding for the enzymes of the PHB biosynthetic pathway, on the synthesis of alkylresorcinols and cyst formation. The phb mutations did not impair the capacity to form mature cysts. However, the cysts formed by these strains posses a thicker exine layer and a higher content of alkylresorcinols than the cysts formed by the wild-type strain. A blockage of PHB synthesis caused by phb mutations resulted in the synthesis of alkylresorcinols and encystment even under non-inducing conditions. We propose that, as a consequence of the blockage in the PHB biosynthetic pathway, the acetyl-CoA and reducing power pools are increased causing the shift to lipid metabolism required for the synthesis of alkylresorcinols and cyst formation.

Keywords: Encystment Polyhydroxyalkanoate PHB synthase

The anabolic pyruvate oxidoreductase from Methanococcus maripaludis by Winston C. Lin; Yu-Ling Yang; William B. Whitman (pp. 444-456).

In autotrophic methanogens, pyruvate oxidoreductase (POR) plays a key role in the assimilation of CO₂ and the biosynthesis of organic carbon. This enzyme has been purified to homogeneity, and the genes from Methanococcus maripaludis were sequenced. The purified POR contained five polypeptides with molecular masses of 47, 33, 25, 21.5 and 13 kDa. The N-terminal sequences of four of the polypeptides had high similarity to the subunits commonly associated with this enzyme from other archaea. However, the 21.5-kDa polypeptide had not been previously observed in PORs. Nucleotide sequencing of the gene cluster encoding the POR revealed six open reading frames (porABCDEF). The genes porABCD corresponded to the subunits previously identified in PORs. On the basis of the N-terminal amino acid sequence, porE encoded the 21.5-kDa polypeptide and contained a high cysteinyl residue content and a motif indicative of a [Fe–S] cluster. porF also had a high sequence similarity to porE, a high cysteinyl residue content, and two [Fe–S] cluster motifs. Homologs to porE were also present in the genomic sequences of the autotrophic methanogens Methanocaldococcus jannaschii and Methanothermobacter thermautotrophicus. Based upon these results, it is proposed that PorE and PorF are components of a specialized system required to transfer low-potential electrons for pyruvate biosynthesis. Some biochemical properties of the purified methanococcal POR were also determined. This unstable enzyme was very sensitive to O₂and demonstrated high activity with pyruvate, oxaloacetate, and α-ketobutyrate. Methyl viologen, rubredoxin, FMN, and FAD were readily reduced. Activity was also observed with spinach and clostridial ferredoxins and cytochrome c. Coenzyme F₄₂₀ was not an electron acceptor for the purified enzyme.

Keywords: Methanococcus maripaludis Pyruvate oxidoreductase Methanogen Archaea

Sporotomaculum syntrophicum sp. nov., a novel anaerobic, syntrophic benzoate-degrading bacterium isolated from methanogenic sludge treating wastewater from terephthalate manufacturing by Yan-Ling Qiu; Yuji Sekiguchi; Hiroyuki Imachi; Yoichi Kamagata; I-Cheng Tseng; Sheng-Shung Cheng; Akiyoshi Ohashi; Hideki Harada (pp. 457-457).

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Archives of Microbiology (v.179, #6)

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Archives of Microbiology (v.179, #6)