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Archives of Microbiology (v.194, #11)

Cyclic lipopeptide profile of the plant-beneficial endophytic bacterium Bacillus subtilis HC8 by Natalia Malfanova; Laurent Franzil; Ben Lugtenberg; Vladimir Chebotar; Marc Ongena (pp. 893-899).

In a previous study (Malfanova et al. in Microbial Biotech 4:523–532, 2011), we described the isolation and partial characterization of the biocontrol endophytic bacterium B. subtilis HC8. Using thin-layer chromatography, we have detected several bioactive antifungal compounds in the methanolic extract from the acid-precipitated supernatant of HC8. In the present study, we have further analyzed this methanolic extract using liquid chromatography-mass spectrometry. Based on the comparison of retention times and molecular masses with those of known antifungal compounds, we identified three families of lipopeptide antibiotics. These include four iturins A having fatty acyl chain lengths of C14 to C17, eight fengycins A (from C14 to C18 and from C15 to C17 containing a double bond in the acyl chain), four fengycins B (C15 to C18), and five surfactins (C12 to C16). Evaluation of the antifungal activity of the isolated lipopeptides showed that fengycins are the most active ones. To our knowledge, this is the first report of an endophytic Bacillus subtilis producing all three major families of lipopeptide antibiotics containing a very heterogeneous mixture of homologues. The questions remain open which of these lipopeptides (1) are being produced during interaction with the plant and (2) are contributing to the biocontrol activity of HC8.

Keywords: Endophytic; Bacillus ; Cyclic lipopeptides; LC–MS

Acetatifactor muris gen. nov., sp. nov., a novel bacterium isolated from the intestine of an obese mouse by Nora Pfeiffer; Charles Desmarchelier; Michael Blaut; Hannelore Daniel; Dirk Haller; Thomas Clavel (pp. 901-907).

We used selective agar media for culturing bacteria from the caecum of mice fed a high calorie diet. In addition to the isolation of Enterobacteriaceae growing on a medium containing cholesterol and bile salts, we focused on the characterization of strain CT-m2^T, which, based on 16S rDNA analysis, did not appear to correspond to any currently described organisms. The isolate belongs to the Clostridium cluster XIV and is most closely related to members of the Lachnospiraceae, including the genera Anaerostipes, Blautia, Butyrivibrio, Clostridium, Coprococcus, Eubacterium, Robinsoniella, Roseburia, Ruminococcus and Syntrophococcus (≤90 % similarity). Strain CT-m2^T is a non-motile Gram-positive rod that does not form spores and has a G + C content of DNA of 48.5 %. Cells grow under strictly anoxic conditions (100 % N₂) and produce acetate and butyrate after growth in reduced WCA broth. In contrast to related species, the new bacterium does not metabolize glucose and is positive for phenylalanine arylamidase, and its major cellular fatty acid is C_14:0. Based on phylogenetic and phenotypic studies, the isolate merits recognition as a member of a novel genus and species, for which the name Acetatifactor muris is proposed. The type strain is CT-m2^T (= DSM 23669^T = ATCC BAA-2170^T).

Keywords: Mouse intestinal microbiota; Diet-induced obesity; Firmicutes ; Clostridia ; Acetatifactor muris

Anabaena sp. PCC7120 transformed with glycine methylation genes from Aphanothece halophytica synthesized glycine betaine showing increased tolerance to salt by Rungaroon Waditee-Sirisattha; Meenakshi Singh; Hakuto Kageyama; Daungjai Sittipol; Ashwani K. Rai; Teruhiro Takabe (pp. 909-914).

Photosynthetic, nitrogen-fixing Anabaena strains play an important role in the carbon and nitrogen cycles in tropical paddy fields although they are salt sensitive. Improvement in salt tolerance of Anabaena cells by expressing glycine betaine–synthesizing genes is an interesting subject. Due to the absence of choline in cyanobacteria, choline-oxidizing enzyme could not be used for the synthesis of glycine betaine. Here, the genes encoding glycine-sarcosine and dimethylglycine methyltransferases (ApGSMT-DMT) from a halotolerant cyanobacterium Aphanothece halophytica were expressed in Anabaena sp. strain PCC7120. The ApGSMT-DMT-expressing Anabaena cells were capable of synthesizing glycine betaine without the addition of any substance. The accumulation level of glycine betaine in Anabaena increased with rise of salt concentration. The transformed cells exhibited an improved growth and more tolerance to salinity than the control cells. The present work provides a prospect to engineer a nitrogen-fixing cyanobacterium having enhanced tolerance to stress by manipulating de novo synthesis of glycine betaine.

Keywords: Aphanothece halophytica ; Betaine; Glycine methylation; Salt tolerance

ABC transporter genes from Streptomyces ghanaensis moenomycin biosynthetic gene cluster: roles in antibiotic production and export by Bohdan Ostash; Emma Doud; Suzanne Walker (pp. 915-922).

Streptomyces ghanaensis ATCC14672 produces antibiotic moenomycin A (MmA), which possesses strong antibacterial activity. The genetic control of MmA biosynthesis has been recently elucidated; nevertheless, little is known about the roles of two pairs of genes, moeX5moeP5 and moeD5moeJ5, coding for ATP-dependent transporter systems. Here we report that both gene pairs form transcriptional units actively expressed during MmA production phase. Streptomyces ghanaensis mutants deficient in either (one) or both transporter systems are characterized by a decreased ability to produce moenomycins, and the ΔmoeP5moeX5 mutant exported less moenomycins. However, even the quadruple S. ghanaensis mutant (ΔmoeD5moeJ5 + ΔmoeX5moeP5) remains able to extrude significant amounts of moenomycin. Similar results were observed under conditions of heterologous expression of moe cluster. Transporter genes other than those located in moe cluster are likely to participate in moenomycin efflux.

Keywords: Streptomyces ghanaensis ; Moenomycin; ABC transporters

Specific PCR-based detection of Alternaria helianthi: the cause of blight and leaf spot in sunflower by A. C. Udayashankar; S. Chandra Nayaka; B. Archana; G. Anjana; S. R. Niranjana; C. N. Mortensen; Ole S. Lund; H. S. Prakash (pp. 923-932).

Alternaria helianthi is an important seed-borne pathogenic fungus responsible for blight disease in sunflower. The current detection methods, which are based on culture and morphological identification, are time-consuming, laborious and are not always reliable. A PCR-based diagnostic method was developed with species-specific primers designed based on the sequence data of a region consisting of the 5.8S RNA gene and internal transcribed spacers—ITS 1 and ITS 2 of nuclear ribosomal RNA gene (rDNA) repeats of A. helianthi. The specificity of the primer pairs AhN1F and AhN1R designed was verified by PCR analysis of DNA from 18 Alternaria helianthi strains isolated from India, 14 non-target Alternaria spp. and 11 fungal isolates of other genera. A single amplification product of 357-bp was detected from DNA of A. helianthi isolates. No cross-reaction was observed with any of the other isolates tested. The detection limit of the PCR method was of 10 pg from template DNA. The primers could also detect the pathogen in infected sunflower seed. This species-specific PCR method provides a quick, simple, powerful and reliable alternative to conventional methods in the detection and identification of A. helianthi. This is the first report of an A. helianthi-specific primer set.

Keywords: Alternaria blight; Seed-borne fungi; Seed health; Molecular diagnosis; PCR; Specific primers; ITS sequences

Differential expression of the transcription factors MarA, Rob, and SoxS of Salmonella Typhimurium in response to sodium hypochlorite: down-regulation of rob by MarA and SoxS by B. Collao; E. H. Morales; F. Gil; R. Polanco; I. L. Calderón; C. P. Saavedra (pp. 933-942).

To survive, Salmonella enterica serovar Typhimurium (S. Typhimurium) must sense signals found in phagocytic cells and modulate gene expression. In the present work, we evaluated the expression and cross-regulation of the transcription factors MarA, Rob, and SoxS in response to NaOCl. We generated strains ΔsoxS and ΔmarA, which were 20 times more sensitive to NaOCl as compared to the wild-type strain; while Δrob only 5 times. Subsequently, we determined that marA and soxS transcript and protein levels were increased while those of rob decreased in a wild-type strain treated with NaOCl. To assess if changes in S. Typhimurium after exposure to NaOCl were due to a cross-regulation, as in Escherichia coli, we evaluated the expression of marA, soxS, and rob in the different genetic backgrounds. The positive regulation observed in the wild-type strain of marA and soxS was retained in the Δrob strain. As in the wild-type strain, rob was down-regulated in the ΔmarA and ΔsoxS treated with NaOCl; however, this effect was decreased. Since rob was down-regulated by both factors, we generated a ΔmarA ΔsoxS strain finding that the negative regulation was abolished, confirming our hypothesis. Electrophoretic mobility shift assays using MarA and SoxS confirmed an interaction with the promoter of rob.

Keywords: marA/rob/soxS regulon; Oxidative stress; Sodium hypochlorite

Genome analysis and heterologous expression of acetate-activating enzymes in the anammox bacterium Kuenenia stuttgartiensis by Lina Russ; Harry R. Harhangi; Jeroen Schellekens; Bram Verdellen; Boran Kartal; Huub J. M. Op den Camp; Mike S. M. Jetten (pp. 943-948).

Anaerobic ammonium-oxidizing bacteria were recently shown to use short-chain organic acids as additional energy source. The AMP-forming acetyl-CoA synthetase gene (acs) of Kuenenia stuttgartiensis, encoding an important enzyme involved in the conversion of these organic acids, was identified and heterologously expressed in Escherichia coli to investigate the activation of several substrates, that is, acetate, propionate and butyrate. The heterologously expressed ACS enzyme could complement an E. coli triple mutant deficient in all pathways of acetate activation. Activity was observed toward several short-chain organic acids, but was highest with acetate. These properties are in line with a mixotrophic growth of anammox bacteria. In addition to acs, the genome of K. stuttgartiensis contained the essential genes of an acetyl-CoA synthase/CO dehydrogenase complex and genes putatively encoding two isoenzymes of archaeal-like ADP-forming acetyl-CoA synthetase underlining the importance of acetyl-CoA as intermediate in the carbon assimilation metabolism of anammox bacteria.

Keywords: Acetate; Propionate; Acetyl-coenzyme A; Anammox; AMP

(R)-Cysteate-nitrogen assimilation by Cupriavidus necator H16 with excretion of 3-sulfolactate: a patchwork pathway by Jutta Mayer; Karin Denger; Klaus Hollemeyer; David Schleheck; Alasdair M. Cook (pp. 949-957).

Cupriavidus necator H16 grew exponentially with (R)-cysteate, a structural analogue of aspartate, as sole source of nitrogen in succinate-salts medium. Utilization of cysteate was quantitative and concomitant with growth and with the excretion of the deaminated product (R)-sulfolactate, which was identified thoroughly. The deaminative pathway started with transport of (R)-cysteate into the cell, which we attributed to an aspartate transporter. Transamination to sulfopyruvate involved an aspartate/(R)-cysteate:2-oxoglutarate aminotransferase (Aoa/Coa) and regeneration of the amino group acceptor by NADP⁺-coupled glutamate dehydrogenase. Reduction of sulfopyruvate to (R)-sulfolactate was catalyzed by a (S)-malate/(R)-sulfolactate dehydrogenase (Mdh/Sdh). Excretion of the sulfolactate could be attributed to the sulfite/organosulfonate exporter TauE, which was co-encoded and co-expressed, with sulfoacetaldehyde acetyltransferase (Xsc), though Xsc was irrelevant to the current pathway. The metabolic enzymes could be assayed biochemically. Aoa/Coa and Mdh/Sdh were highly enriched by protein separation, partly characterized, and the relevant locus-tags identified by peptide-mass fingerprinting. Finally, RT-PCR was used to confirm the transcription of all appropriate genes. We thus demonstrated that Cupriavidus necator H16 uses a patchwork pathway by recruitment of ‘housekeeping’ genes and sulfoacetaldehyde-degradative genes to scavenge for (R)-cysteate-nitrogen.

Keywords: Sulfonate metabolism; Cysteate aminotransferase; 3-sulfopyruvate dehydrogenase; Sulfolactate excretion; Transport

Roles of DNA repair and membrane integrity in heat resistance of Deinococcus radiodurans by Anja Bauermeister; Claudia Hahn; Petra Rettberg; Günther Reitz; Ralf Moeller (pp. 959-966).

To study the effects of heat shock on Deinococcus radiodurans and the role of DNA repair in high temperature resistance, different strains of D. radiodurans (wild type, recA, irrE, and pprA) were treated with temperatures ranging from 40 to 100 °C under wet and dry conditions. The mutant strains were more sensitive to wet heat of ≥60 °C and dry heat of ≥80 °C than the wild type. Both wild-type and DNA repair-deficient strains were much more resistant to high temperatures when exposed in the dried state as opposed to cells in suspension. Molecular staining techniques with the wild-type strain revealed that cells in the dried state were able to retain membrane integrity after drying and subsequent heat exposure, while heat-exposed cells in suspension showed significant loss of membrane integrity and respiration activity. The results suggest that the repair of DNA damage (e.g., DNA double-strand breaks by RecA and PprA) is essential after treatment with wet heat at temperatures >60 °C and dry heat >80 °C, and the ability of D. radiodurans to stabilize its plasma membrane during dehydration might represent one aspect in the protection of dried cells from heat-induced membrane damage.

Keywords: Deinococcus radiodurans ; Heat resistance; DNA damage; Membrane lesions; DNA repair mechanisms

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Archives of Microbiology (v.194, #11)

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Archives of Microbiology (v.194, #11)