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Archives of Microbiology (v.191, #4)

Genetic analysis of the role of the ABC transporter Ota and Otb in glycine betaine transport in Methanosarcina mazei Gö1 by Regina Saum; Ana Mingote; Helena Santos; Volker Müller (pp. 291-301).

The methanogenic archaeon Methanosarcina mazei Gö1 accumulates glycine betaine in response to hypersalinity but differs from most other methanoarchaea in having two gene clusters both encoding a potential glycine betaine transporter, Ota and Otb. We have created mutants with either ota or otb deleted to address their role in salt adaptation. The mutants were not impaired in growth at low or high salt, neither at 37°C nor at lower temperatures. However, the ∆ota mutant was completely defective in glycine betaine transport demonstrating that Ota is the only glycine betaine transporter in M. mazei. The mutation in otb led to increased transcription of ota and thus increased transport and accumulation of glycine betaine suggesting a cross talk between the two transporters.

Keywords: Archaea; Methanogens; Methanosarcina mazei Gö1; Salt adaptation; Betaine transport

Physico-chemical characterization of a new heteropolysaccharide produced by a native isolate of heterofermentative Lactobacillus sp. CFR-2182 by S. V. N. Vijayendra; G. Palanivel; S. Mahadevamma; R. N. Tharanathan (pp. 303-310).

A heterofermentative Lactobacillus sp. CFR-2182 was isolated from dahi samples and it was found to produce 8.0 and 20.5 g/L heteropolysaccharide (HePS) in EPS medium (a simplified synthetic medium) and modified MRS broth, respectively, after 72 h at 30°C. The total carbohydrate, reducing sugar and moisture contents of the purified HePS were 74, 10.6 and 2 g, respectively, per 100 g on dry weight basis. The HePS produced in EPS medium had glucose and mannose in 17:1 ratio. The HePS was non-gelling and non-film forming type. It was completely soluble in water and 1 N sodium hydroxide solution. Gel permeation chromatography and HPLC analysis indicated considerable heterogeneity of the HePS, having three fractions with molecular weights ranging from 3.3 × 10⁴ to 1.32 × 10⁶ Da. The enzymatic hydrolysis of the HePS with pullulanase and α-amylase [with α(1→4) linkage] indicated the presence of α(1→6) and traces of α(1→4) linkages, respectively. NMR analysis of the EPS revealed unique chemical shifts.

Keywords: Chromatography; EPS medium; Fermentation; Heteropolysaccharide; Lactobacillus

Root nodule bacteria isolated from South African Lotononis bainesii, L. listii and L. solitudinis are species of Methylobacterium that are unable to utilize methanol by Julie Kaye Ardley; Graham W. O’Hara; Wayne G. Reeve; Ron J. Yates; Michael J. Dilworth; Ravi P. Tiwari; John G. Howieson (pp. 311-318).

The South African legumes Lotononis bainesii, L. listii and L. solitudinis are specifically nodulated by highly effective, pink-pigmented bacteria that are most closely related to Methylobacterium nodulans on the basis of 16S rRNA gene homology. Methylobacterium spp. are characterized by their ability to utilize methanol and other C₁ compounds, but 11 Lotononis isolates neither grew on methanol as a sole carbon source nor were able to metabolize it. No product was obtained for PCR amplification of mxaF, the gene encoding the large subunit of methanol dehydrogenase. Searches for methylotrophy genes in the sequenced genome of Methylobacterium sp. 4-46, isolated from L. bainesii, indicate that the inability to utilize methanol may be due to the absence of the mxa operon. While methylotrophy appears to contribute to the effectiveness of the Crotalaria/M. nodulans symbiosis, our results indicate that the ability to utilize methanol is not a factor in the Lotononis/Methylobacterium symbiosis.

Keywords: Methylobacterium ; Lotononis ; Methylotrophy; Root nodule bacteria

A small heat-shock protein confers stress tolerance and stabilizes thylakoid membrane proteins in cyanobacteria under oxidative stress by Kollimalai Sakthivel; Tatsuro Watanabe; Hitoshi Nakamoto (pp. 319-328).

Small heat-shock proteins are molecular chaperones that bind and prevent aggregation of nonnative proteins. They also associate with membranes. In this study, we show that the small heat-shock protein HspA plays a protective role under oxidative stress in the cyanobacterium Synechococcus elongatus strain ECT16-1, which constitutively expresses HspA. Compared with the reference strain ECT, ECT16-1 showed much better growth and viability in the presence of hydrogen peroxide. Under the peroxide stress, pigments in thylakoid membrane, chlorophyll, carotenoids, and phycocyanins, were continuously reduced in ECT, but in ECT16-1 they decreased only during the first 24 h of stress; thereafter no further reduction was observed. For comparison, we analyzed a wild type and an hspA deletion strain from Synechocystis sp. PCC 6803 and found that lack of hspA significantly affected the viability of the cell and the pigment content in the presence of methyl viologen, suggesting that HspA stabilizes membrane proteins such as the photosystems and phycobilisomes from oxidative damage. In vitro pull down assays showed a direct interaction of HspA with components of phycobilisomes. These results show that HspA and small heat-shock proteins in general play an important role in the acclimation to oxidative stress in cyanobacteria.

Keywords: Cyanobacterium; Oxidative stress; Photosystem; Phycobilisome; Phycocyanin; Small heat-shock protein

Characterization of diverse heterocyclic amine-degrading denitrifying bacteria from various environments by Hee-Sung Bae; Wan-Taek Im; Yuichi Suwa; James M. Lee; Sung-Taik Lee; Young-Keun Chang (pp. 329-340).

Although, there have been many published bacterial strains aerobically degrading the heterocyclic amine compounds, only one strain to date has been reported to degrade pyrrolidine under denitrifying conditions. In this study, denitrifying bacteria degrading pyrrolidine and piperidine were isolated from diverse geological and ecological origins through selective enrichment procedures. Based on the comparative sequence results of 16S rRNA genes, 30 heterocyclic amine-degrading isolates were grouped into ten distinct phylotypes belonging to the genera Thauera, Castellaniella, Rhizobium, or Paracoccus of the phylum Proteobacteria. The representative isolates of individual phylotypes were characterized by phylogenetic, phenotypic and chemotaxonomical traits, and dissimilatory nitrite reductase gene (nirK and nirS). All isolates completely degraded pyrrolidine and piperidine under both aerobic and anaerobic conditions. The anaerobic degradations were coupled to nitrate reduction. A metabolic pathway for the anaerobic degradation of pyrrolidine was proposed on the basis of enzyme activities implicated in pyrrolidine metabolism from three isolates. The three key pyrrolidine-metabolizing enzymes pyrrolidine dehydrogenase, γ-aminobutyrate/α-ketoglutarate aminotransferase, and succinic semialdehyde dehydrogenase, were induced by heterocyclic amines under denitrifying conditions. They were also induced in cells grown aerobically on heterocyclic amines, suggesting that the anaerobic degradation of pyrrolidine shares the pathway with aerobic degradation.

Keywords: Heterocyclic amine; Anaerobic degradation; Denitrification; Pyrrolidine metabolism

A new Tunisian strain of Bacillus thuringiensis kurstaki having high insecticidal activity and δ-endotoxin yield by Imen Saadaoui; Souad Rouis; Samir Jaoua (pp. 341-348).

BLB1 is a new Bacillus thuringiensis kurstaki strain, isolated from a Tunisian soil sample. Assay of toxicity of BLB1 crystal proteins resulted in an LC50 of 70.32 ng of toxin per mg of flour against third instar Ephestia kuehniella with confidence limits of (31.6–109.04 ng). This LC50 is less than that of the commercial strains HD1 used as a reference. The characterization of this strain by scanning transmission electron microscopy, analysis of its cry genes content by PCR-sequencing, and analysis of its δ-endotoxin patterns demonstrate that it belongs to the same subgroup than HD1, but ruled out the involvement of cry gene content or protoxin activation in the hypertoxicity of this strain. Taking into account the δ-endotoxin/spore ratio for each strain, and by allowing the estimation of the production level per spore, it might be concluded that BLB1 production is the highest, when compared with that of HD1. On the basis of its toxicity, BLB1 could be considered as a strain of great interest and would allow the production of quantities of bioinsecticides at low cost.

Keywords: Bacillus thuringiensis ; Cry protein; HD1; Ephestia kuehniella ; Hypertoxicity

Artificial sRNAs activating the Gac/Rsm signal transduction pathway in Pseudomonas fluorescens by Claudio Valverde (pp. 349-359).

In Pseudomonas fluorescens CHA0, the synthesis of antifungal compounds is post-transcriptionally activated by the Gac/Rsm cascade. The two-component system GacS/GacA promotes transcription of three small regulatory RNAs (i.e., sRNAs), RsmX, RsmY, and RsmZ, which remove the regulatory proteins RsmA and RsmE from the ribosome-binding sites of exoproduct-related mRNAs. The GacS/GacA-dependent accumulation of RsmX/Y/Z and formation of RsmX/Y/Z-RsmA/E complexes relieve mRNA translational repression. Other bacteria as E. coli and Vibrio spp. utilize similar sRNA–protein based systems to adjust mRNA translation (e.g., the E. coli Csr system for carbon storage, motility and biofilm regulation). The Rsm/Csr sRNAs are remarkably similar in that they contain several stem-loops with an invariant GGA trinucleotide exposed in the hairpin loop that would be the characteristic structural-sequence motifs relevant for sRNA activity and stability. Here it is shown that the dysfunctional Gac/Rsm cascade of P. fluorescens ΔrsmXYZ mutants could be restored by appropriate transcription levels of artificial genes encoding RNAs with unrelated primary sequence but with two or more hairpins displaying the RsmA/E binding motifs. The results support the hypothesis that the molecular mimicry of Rsm/Csr sRNAs is based on proper secondary structures that expose critical binding motifs irrespective of their overall sequence.

Keywords: Pseudomonas fluorescens ; Riboregulation; Molecular mimicry; Gac/Rsm cascade; Artificial sRNAs

Quinoprotein ethanol dehydrogenase from Pseudomonas aeruginosa: the unusual disulfide ring formed by adjacent cysteine residues is essential for efficient electron transfer to cytochrome c ₅₅₀ by Bina Mennenga; Christopher W. M. Kay; Helmut Görisch (pp. 361-367).

All pyrroloquinoline quinone (PQQ)-dependent alcohol dehydrogenases contain an unusual disulfide ring formed between adjacent cysteine residues. A mutant enzyme that is lacking this structure was generated by replacing Cys105 and Cys106 with Ala in quinoprotein ethanol dehydrogenase (QEDH) from Pseudomonas aeruginosa ATCC17933. Heterologously expressed quinoprotein ethanol dehydrogenase in which Cys-105 and Cys-106 have been replaced by Ala (Cys105Ala/Cys106Ala apo-QEDH) was successfully converted to enzymatic active holo-enzyme by incorporation of its cofactor PQQ in the presence of Ca²⁺. The enzymatic activity of the mutant enzyme in the artificial dye test with N-methylphenazonium methyl sulfate (PMS) and 2,6-dichlorophenol indophenol (DCPIP) at pH 9 did not depend on an activating amine which is essential for wild type activity under these conditions. The mutant enzyme showed increased Michaelis constants for primary alcohols, while the affinity for the secondary alcohol 2-propanol was unaltered. Surprisingly, for all substrates tested the specific activity of the mutant enzyme in the artificial dye test was higher than that found for wild type QEDH. On the contrary, in the ferricyanide test with the natural electron acceptor cytochrome c ₅₅₀ the activity of mutant Cys105Ala/Cys106Ala was 15-fold lower than that of wild type QEDH. We demonstrate for the first time unambiguously that the unusual disulfide ring is essential for efficient electron transfer at pH 7 from QEDH to its natural electron acceptor cytochrome c ₅₅₀.

Keywords: Active site; Adjacent Cys/Ala replacement; Pyrroloquinoline quinone (PQQ); QEDH; Site directed mutagenesis

Exopolysaccharide from surface-liquid culture of Clonostachys rosea originates from autolysis of the biomass by Graciele Viccini; Thalita Romano Martinelli; Regielly Caroline Raimundo Cognialli; Rodrigo Otávio de Faria; Elaine Rosechrer Carbonero; Guilherme Lanzi Sassaki; David Alexander Mitchell (pp. 369-378).

We describe the purification and chemical characterization of galactomannans that appear both in the biomass and the culture broth during surface-liquid culture of the fungus Clonostachys rosea, a common facultative saprophyte that has potential to be used as a biological control agent against several plant pathogenic fungi, insects and nematodes. The galactomannans from both sources had comparable ratios of Man, Gal and Glc and the similarity were confirmed by ¹H, ¹³C NMR, HMQC, and COSY spectra. We propose that the galactomannan in the culture broth originates from autolysis of the biomass, based not only on the similarity that it has with the galactomannan extracted from the biomass but also on the fact that its concentration increased rapidly after glucose depletion from the medium, when biomass concentration was falling. Polysaccharides from C. rosea have not previously been characterized; we show that the characteristics of the galactomannans are consistent with those that have been reported for other members of the Bionectriaceae, the family to which C. rosea belongs.

Keywords: Galactomannan; Bionectriaceae; NMR spectroscopy; Gliocladium roseum ; Polysaccharides

Proteomic identification of CBM37-containing cellulases produced by the rumen cellulolytic bacterium Ruminococcus albus 20 and their putative involvement in bacterial adhesion to cellulose by Harivony Rakotoarivonina; Cécile Terrie; Christophe Chambon; Evelyne Forano; Pascale Mosoni (pp. 379-388).

The objective of this study was to identify and characterize other proteins than fimbrial proteins potentially involved in R. albus 20 adhesion to cellulose using an adhesion-related antiserum preparation (i.e. anti-Adh serum). From protein fractions of R. albus 20 grown on cellulose, the serum recognized at least 10 cellulose-binding proteins (CBPs), among which homologs of glycoside hydrolases (family 5, 9 and 48) of R. albus 8 (i.e. Cel5G, Cel9B and Cel48A) were identified by a proteomic approach. In strain 20, Cel9B and Cel48A were identified as two major CBPs and as bacterial cell-associated proteins. The anti-Adh serum was also shown to target the C-terminal family 37 carbohydrate-binding module (CBM37) of Cel9B and Cel48A, indicating that this module, unique to R. albus, may play a significant role in bacterial adhesion to cellulose as suggested previously for R. albus 8. Overall, our results support the hypothesis of an adhesion mechanism involving the CBM37 of Cel9B and Cel48A. This adhesion mechanism may not be restricted to these two enzymes but may also involve other CBM37-containing proteins such as Cel5G and the other uncharacterised proteins recognized by the anti-Adh serum.

Keywords: Ruminococcus albus ; Two-dimensional gel electrophoresis; MALDI-TOF mass spectrometry; Cellulolysis; Adhesion

Stimulation of indoleacetic acid production in a Rhizobium isolate of Vigna mungo by root nodule phenolic acids by Santi Mandal; Mahitosh Mandal; Amit Das; Bikas Pati; Ananta Ghosh (pp. 389-393).

The influence of endogenous root nodules phenolic acids on indoleacetic acid (IAA) production by its symbiont (Rhizobium) was examined. The root nodules contain higher amount of IAA and phenolic acids than non-nodulated roots. Presence of IAA metabolizing enzymes, IAA oxidase, peroxidase, and polyphenol oxidase indicate the metabolism of IAA in the nodules and roots. Three most abundant endogenous root nodule phenolic acids (protocatechuic acid, 4-hydroxybenzaldehyde and p-coumaric acid) have been identified and their effects on IAA production by the symbiont have been studied in l-tryptophan supplemented yeast extract basal medium. Protocatechuic acid (1.5 μg ml⁻¹) showed maximum stimulation (2.15-fold over control) of IAA production in rhizobial culture. These results indicate that the phenolic acids present in the nodule might serve as a stimulator for IAA production by the symbiont (Rhizobium).

Keywords: IAA; Phenolic acids; Rhizobium sp.; Root nodule; Vigna mungo

Stimulation of indoleacetic acid production in a Rhizobium isolate of Vigna mungo by root nodule phenolic acids by Santi M. Mandal; Mahitosh Mandal; Amit K. Das; Bikas R. Pati; Ananta K. Ghosh (pp. 395-395).

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Archives of Microbiology (v.191, #4)