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Archives of Microbiology (v.188, #6)
Paired cloning vectors for complementation of mutations in the cyanobacterium Anabaena sp. strain PCC 7120 by C. Peter Wolk; Qing Fan; Ruanbao Zhou; Guocun Huang; Sigal Lechno-Yossef; Tanya Kuritz; Elizabeth Wojciuch (pp. 551-563).
The clones generated in a sequencing project represent a resource for subsequent analysis of the organism whose genome has been sequenced. We describe an interrelated group of cloning vectors that either integrate into the genome or replicate, and that enhance the utility, for developmental and other studies, of the clones used to determine the genomic sequence of the cyanobacterium, Anabaena sp. strain PCC 7120. One integrating vector is a mobilizable BAC vector that was used both to generate bridging clones and to complement transposon mutations. Upon addition of a cassette that permits mobilization and selection, pUC-based sequencing clones can also integrate into the genome and thereupon complement transposon mutations. The replicating vectors are based on cyanobacterial plasmid pDU1, whose sequence we report, and on broad-host-range plasmid RSF1010. The RSF1010- and pDU1-based vectors provide the opportunity to express different genes from either cell-type-specific or -generalist promoters, simultaneously from different plasmids in the same cyanobacterial cells. We show that pDU1 ORF4 and its upstream region play an essential role in the replication and copy number of pDU1, and that ORFs alr2887 and alr3546 (hetF A ) of Anabaena sp. are required specifically for fixation of dinitrogen under oxic conditions.
Keywords: Anabaena sp. strain PCC 7120; Cloning vectors; Complementation of mutations
Spontaneous zygogenesis (Z-mating) in mecillinam-rounded bacteria by Jean-Pierre Gratia (pp. 565-574).
In Escherichia coli the process of spontaneous zygogenesis (Z-mating), i.e. complete genetic mixing in the absence of a conjugative plasmid, was investigated further. Spontaneous-zygogenesis-promoting (Szp+) cells displayed strong clustering with each other and with ordinary F− cells in the optimal cell density range for Z-mating. When induced to rounding by the drug mecillinam, they aggregated into large, dense, stainable syncytium-like cells leaving giant ghosts upon lysis. In Z-mating mixtures of mecillinam-treated cells, these giant cells co-purified with mating products as other cells died. Giant cells recovering from mecillinam treatment yielded monstrous, branching forms, whereas non-Szp+ coccal cells reverted to rods in one step, and some 29% of the colonies formed were identified as deriving from entities possessing two distinct genomes. Z-mating was examined between E. coli and a distantly related Serratia marcescens strain. In the presence of calcium, mecillinam-rounded cells of a stable non-complementing diploid hybrid with the E. coli phenotype segregated normally dividing cells of the Serratia form.
Keywords: Bacterial cell clustering; Complete zygote formation; Escherichia coli ; Mecillinam; Serratia marcescens ; Syncytium-like bacterial cells
Identification and characterization of sawC, a whiA-like gene, essential for sporulation in Streptomyces ansochromogenes by Zhoujie Xie; Wenli Li; Yuqing Tian; Gang Liu; Huarong Tan (pp. 575-582).
sawC, encoding a protein homologous to the WhiA sporulation regulator of Streptomyces coelicolor, was cloned from a fairly distantly related species, Streptomyces ansochromogenes. Disruption of sawC led to formation of aerial mycelium much longer than normal spore chains and with somewhat increased coiling, indicating that sawC plays an important role in the cessation of aerial hyphal growth similar to that of whiA, and that it has an effect on cell wall structure. However, complementation of sawC and whiA mutants with the same DNA fragment gave different results, which suggested that there may be some difference in regulation or function of WhiA/SawC between the two strains. S1 nuclease mapping identified one transcription start point of sawC. Using EGFP as a reporter, the spatial expression of sawC was shown to be confined to aerial hyphae. Computer-aided structural prediction analysis of SawC/WhiA proteins revealed the presence of a fold very similar to the endonuclease domain of PI-PfuI, raising the possibility that these proteins may interact with an intermediate in DNA repair or replication.
Keywords: sawC ; whiA ; Morphological differentiation; Strptomyces ansochromogenes
Anaerobic growth of Escherichia coli on d-tartrate depends on the fumarate carrier DcuB and fumarase, rather than the l-tartrate carrier TtdT and l-tartrate dehydratase by Ok Bin Kim; Sebastian Lux; Gottfried Unden (pp. 583-589).
Escherichia coli is able to grow under anaerobic conditions on d-tartrate when glycerol is supplied as an electron donor (d-tartrate fermentation). d-Tartrate was converted to succinate. Growth was lost in strains deficient for DcuB, the fumarate/succinate antiporter of fumarate respiration. The l-tartrate/succinate antiporter TtdT of l-tartrate fermentation, or the C4-dicarboxylate carriers DcuA and DcuC, were not able to support d-tartrate transport and fermentation. Deletion of fumB demonstrated, that fumarase B is required for growth on d-tartrate. The mutant lost most (about 79%) of d-tartrate dehydratase activity. l-Tartrate dehydratase (TtdAB), and fumarase A or C, showed no or only a small contribution to d-tartrate dehydratase activity. Therefore d-tartrate is metabolised by a sequence of reactions analogous to that from l-tartrate fermentation, including dehydration to oxaloacetate, which is then converted to malate, fumarate and succinate. The stereoisomer specific carrier TtdT and dehydratase TtdAB of l-tartrate fermentation are substituted by enzymes from general anaerobic fumarate metabolism, the antiporter DcuB and fumarase B, which have a broader substrate specificity. No d-tartrate specific carriers and enzymes are involved in the pathway.
Keywords: d-Tartrate fermentation; l-Tartrate fermentation; Fumarate respiration; DcuB; Tartrate carrier; Fumarase; Tartrate dehydratase
Comparison of invasion of fibroblasts and macrophages by high- and low-virulence Leptospira strains: colonization of the host-cell nucleus and induction of necrosis by the virulent strain by Liwei Li; David M. Ojcius; Jie Yan (pp. 591-598).
The infection cycle of low- and high-virulence strains of Leptospira interrogans was compared in fibroblasts and macrophages. L. interrogans serovar Lai strain Lai was used as a representative high-virulence strain, while L. interrogans serovars Pomona strain Luo was used as a low-virulence strain. L. biflexa serovar Patoc strain Patoc I, a nonparasitic strain of Leptospira, was used as a control. Both the high- and low-virulence strains could adhere to fibroblasts and macrophages using one or both ends of the spirochete, which was followed by phagocytosis of both strains. Both strains adhered more strongly to macrophages than fibroblasts. However, the high-virulence strain could invade the host-cell nucleus, while the low-virulence strain remained in phagosomes. The L. biflexa strain neither adhered to nor invaded either cell type. Both of the L. interrogans strains also induced cell death (mostly necrosis) of macrophages, whether or not the spirochetes were viable, suggesting that leptospiral virulence is unrelated to macrophage death. However, the high-virulence strain induced mainly necrosis in fibroblasts, while the low-virulence strain induced more apoptosis. Thus, the main feature distinguishing the two L. interrogans strains is the ability of the high-virulence strain to invade the host-cell nucleus and induce pro-inflammatory necrosis in fibroblasts.
Keywords: Leptospira; Fibroblast; Macrophage; Invasion; Virulence; Nucleus
Rhodobacter capsulatus magnesium chelatase subunit BchH contains an oxygen sensitive iron–sulfur cluster by Nick Sirijovski; Fikret Mamedov; Ulf Olsson; Stenbjörn Styring; Mats Hansson (pp. 599-608).
Magnesium chelatase is the first unique enzyme of the bacteriochlorophyll biosynthetic pathway. It consists of three subunits (BchI, BchD, and BchH). Amino acid sequence analysis of the Rhodobacter capsulatus BchH revealed a novel cysteine motif (393CX2CX3CX14C) that was found in only six other proteobacteria (CX2CX3CX11–14C). The cysteine motif is likely to coordinate an unprecedented [Fe–S] cluster. Purified BchH demonstrated absorbance in the 460 nm region. This absorbance was abolished in BchH proteins with alanine substitutions at positions Cys396 and Cys414. These modified proteins were also EPR silent. In contrast, wild type BchH protein in the reduced state showed EPR signals resembling those of a [4Fe–4S] cluster with rhombic symmetry and g values at 1.90, 1.93, and 2.09, superimposed with a [3Fe–4S] cluster centered at g = 2.02. The [3Fe–4S] signal was observed independently of the [4Fe–4S] signal under oxidizing conditions. Mg-chelatase activity assays showed that the cluster is not catalytic. We suggest that the [4Fe–4S] and [3Fe–4S] signals originate from a single coordination site on the monomeric BchH protein and that the [4Fe–4S] cluster is sensitive to oxidation. It is speculated that the cluster participates in the switching between aerobic and anaerobic life of the proteobacteria.
Keywords: Bacteriochlorophyll; chlH ; Chlorophyll; Tetrapyrrole; Xantha-f ; Enzyme: Magnesium chelatase EC 6.6.1.1
Transcription and regulation of the hydrogenase(s) accessory genes, hypFCDEAB, in the cyanobacterium Lyngbya majuscula CCAP 1446/4 by Daniela Ferreira; Elsa Leitão; Johannes Sjöholm; Paulo Oliveira; Peter Lindblad; Pedro Moradas-Ferreira; Paula Tamagnini (pp. 609-617).
Lyngbya majuscula CCAP 1446/4 is a filamentous cyanobacterium possessing both an uptake and a bi-directional hydrogenase. The presence of a single copy of the hyp operon in the cyanobacterial genomes suggests that these accessory genes might be responsible for the maturation of both hydrogenases. We investigated the concomitant transcription of hypFCDEAB with the hydrogenases structural genes—hup and hox. RT-PCRs performed with L. majuscula cells grown under different physiological conditions showed a substantial decrease in the relative amount of hupL transcript under non-N2-fixing conditions. In contrast, no significant differences were observed for the transcript levels of hypFCDEAB in all conditions tested, while minor fluctuations could be discerned for hoxH. Previously, it was demonstrated that the transcriptional regulators NtcA and LexA interact with the promoter regions of hup and hox, respectively, and that putative binding sites for both proteins are present in the hyp promoter of L. majuscula. Therefore, a putative involvement of NtcA and LexA in the regulation of the hyp transcription was investigated. Electrophoretic mobility shift assays resulted in NtcA or LexA-bound retarded fragments, suggesting the involvement of these proteins in the transcriptional regulation of hypFCDEAB.
Keywords: Lyngbya majuscula ; hyp ; Hydrogenase accessory genes; Transcriptional regulator factors; NtcA; LexA
The regulator CdsS/CdsR two-component system modulates expression of genes involved in chitin degradation of Pseudoalteromonas piscicida strain O-7 by Katsushiro Miyamoto; Mina Okunishi; Eiji Nukui; Takahiro Tsuchiya; Takeshi Kobayashi; Chiaki Imada; Hiroshi Tsujibo (pp. 619-628).
Pseudoalteromonas piscicida strain O-7 (formerly Alteromonas sp. strain O-7) is an efficient degrader of chitin in the marine environment. The chitinolytic system of the strain consists of many enzymes induced by N-acetylglucosamine (GlcNAc). This paper reports that CdsR, which is a response regulator of CdsS/CdsR two-component signal transduction system, is bound to near the promoter region of GlcNAc-induced aprIV gene. The CdsR protein as a response regulator was transphosphorylated by the CdsS protein as a sensor kinase. Furthermore, the transphosphorylation from CdsS to CdsR was promoted by chitin degradation products and a metabolite. The CdsR protein was also phosphorylated by acetyl phosphate which is an indicator of nutritive conditions of cells. Gel mobility shift assays demonstrated that phosphorylated CdsR (CdsR-P) was bound to not only near the promoter region of aprIV gene but also those of chiA, chiB, chiC, chiD and cbp1 genes which are induced in the presence of GlcNAc. Footprinting analysis demonstrated that CdsR-P was bound to the sequences around the transcriptional start sites of aprIV and chiD genes. These results indicate that CdsR is one of the common regulators of these genes involved in chitin degradation of the strain.
Keywords: Chitin; Degradation; Response regulator; Sensor; Chitinase; Protease; Marine; Alteromonas
Structural characterization of diabolic acid-based tetraester, tetraether and mixed ether/ester, membrane-spanning lipids of bacteria from the order Thermotogales by Jaap S. Sinninghe Damsté; W. Irene C. Rijpstra; Ellen C. Hopmans; Stefan Schouten; Melike Balk; Alfons J. M. Stams (pp. 629-641).
The distribution of core lipids in the membranes of nine different species of the order Thermotogales, one of the early and deep branching lineages in the Bacteria, were examined by HPLC/MS and demonstrated to consist of membrane-spanning diglycerol lipids comprised of diabolic acid-derived alkyl moieties. In the Thermotoga species the core membrane lipids are characterized by the presence of both ester and ether bonds, whereas in the phylogenetically more distinct Thermosipho and Fervidobacterium spp. only ester bonds occur. A tentative biosynthetic route for the biosynthesis of these membrane-spanning lipids is proposed. Since species of the order Thermotogales are assumed to have occurred early during the evolution of life on Earth, as suggested by its position in the phylogenetic tree of life, these data suggest that the ability to produce both ether and ester glycerol membrane lipids developed relatively early during microbial evolution.
Keywords: Diabolic acid; Ether lipids; Ester lipids; Thermotoga ; Thermosipho ; Fervidobacterium
Larvicidal activities against agricultural pests of transgenic Escherichia coli expressing combinations of four genes from Bacillus thuringiensis by Vadim Khasdan; Maria Sapojnik; Arieh Zaritsky; A. Rami Horowitz; Sammy Boussiba; Mario Rippa; Robert Manasherob; Eitan Ben-Dov (pp. 643-653).
The genes cry1Ac and cry1Ca from Bacillus thuringiensis subsps. kurstaki HD-73 and aizawai 4J4, respectively, encoding δ-endotoxins against lepidopteran larvae were isolated, cloned and expressed in Escherichia coli, with and without cyt1Aa (encoding cytolytic protein) and p20 (accessory protein) from subsp. israelensis. Nine combinations of the genes under control of an early T7, P A1 inducible promoter, produced the encoding proteins. Toxicities were examined against larvae of three major agricultural pests: Pectinophora gossypiella, Helicoverpa armigera and Spodoptera littoralis. The clones expressing cyt1Aa, with or without p20, were not toxic. The clone expressing cry1Ac (pBt-1A) was the most toxic to P. gossypiella (LC50 of 0.27 × 108 cells g−1). Clone pBt-1CA expressing cry1Ca and cry1Ac displayed the highest toxicity (LC50 of 0.12 × 108 cells ml−1) against S. littoralis. Clone pBt-1CARCy expressing all four genes (cry1Ca, cry1Ac, p20, cyt1Aa) in tandem exhibited the highest toxicity to H. armigera (LC50 of 0.16 × 108 cells ml−1). Cyt1Aa failed to raise the toxicity of these Cry toxins against P. gossypiella and S. littoralis but significantly enhanced toxicity against H. armigera. Two additional clones expressing either cry1Ac or cry1Ca under tandem promoters, P A1 and P psbA (constitutive), displayed significantly higher toxicities (7.5- to 140-fold) than their counterparts with P A1 alone, reducing the LC50 values to below 107 cells ml−1.
Keywords: Bacillus thuringiensis ; δ-Endotoxin; Transgenic Escherichia coli ; Agriculture pests
Orchid-associated bacteria produce indole-3-acetic acid, promote seed germination, and increase their microbial yield in response to exogenous auxin by Elena A. Tsavkelova; Tatiana A. Cherdyntseva; Svetlana Yu. Klimova; Andrey I. Shestakov; Svetlana G. Botina; Alexander I. Netrusov (pp. 655-664).
Germination of orchid seeds is a complex process. In this paper we focus on interactions between the host-plant and its bacterial partners via indole-3-acetic acid (IAA). Originally isolated from the roots of the epiphytic orchid Dendrobium moschatum, the strains of Rhizobium, Microbacterium, Sphingomonas, and Mycobacterium genera were among the most active IAA producers. Addition of exogenous tryptophan significantly enhanced auxin formation both in mineral and complex media. The presence of IAA and indole-3-acetaldehyde was confirmed by HPLC. Indole-3-pyruvic and indole-3-lactic acids were also detected in supernatants of culture filtrates of Sphingomonas sp., Rhizobium sp., and Microbacterium sp., while indole-3-acetamide was identified only in Mycobacterium sp. Some concentration- and strain-dependent effects of exogenous IAA on bacterial development were also established. Treatment of the cultures with 10 and 100 μg/ml of auxin resulted in an increase in microbial yield. None of the investigated strains was able to utilize IAA as a source of carbon and energy. Furthermore, inoculation of D. moschatum seeds with Sphingomonas sp. and Mycobacterium sp. resulted in considerable enhancement of orchid seeds germination. This growth-promoting activity was observed in the absence of any plant growth stimulators or mycorrhizal fungi, usually required for orchid germination.
Keywords: Associative bacteria; Indole-3-acetic acid; Orchids; Seed germination
Thiocapsa imhoffii, sp. nov., an alkaliphilic purple sulfur bacterium of the family Chromatiaceae from Soap Lake, Washington (USA) by Marie Asao; Shinichi Takaichi; Michael T. Madigan (pp. 665-675).
An alkaliphilic purple sulfur bacterium, strain SC5, was isolated from Soap Lake, a soda lake located in east central Washington state (USA). Cells of strain SC5 were gram-negative, non-motile, and non-gas vesiculate cocci, often observed in pairs or tetrads. In the presence of sulfide, elemental sulfur was deposited internally. Liquid cultures were pink to rose red in color. Cells contained bacteriochlorophyll a and spirilloxanthin as major photosynthetic pigments. Internal photosynthetic membranes were of the vesicular type. Optimal growth of strain SC5 occurred in the absence of NaCl (range 0–4%), pH 8.5 (range pH 7.5–9.5), and 32°C. Photoheterotrophic growth occurred in the presence of sulfide or thiosulfate with only a limited number of organic carbon sources. Growth factors were not required, and cells could fix N2. Dark, microaerobic growth occurred in the presence of both an organic carbon source and thiosulfate. Sulfide and thiosulfate served as electron donors for photoautotrophy, which required elevated levels of CO2. Phylogenetic analysis placed strain SC5 basal to the clade of the genus Thiocapsa in the family Chromatiaceae with a 96.7% sequence similarity to its closest relative, Thiocapsa roseopersicina strain 1711T (DSM217T). The unique assemblage of physiological and phylogenetic properties of strain SC5 defines it as a new species of the genus Thiocapsa, and we describe strain SC5 herein as Tca. imhoffii, sp. nov.
Keywords: Anoxygenic phototrophic bacteria; Purple sulfur bacteria; Chromatiaceae ; Thiocapsa ; Alkaliphiles; Extreme environments; Soap Lake; Soda lakes
Small chromosomal integration site of classical CTX prophage in Mozambique Vibrio cholerae O1 biotype El Tor strain by Bhabatosh Das; Kalpataru Halder; Partha Pal; Rupak K. Bhadra (pp. 677-683).
An unusual strain of Vibrio cholerae O1 biotype El Tor harbouring multiple tandem copies of classical CTX prophage caused a cholera epidemic in Mozambique in 2004. However, the location of the classical CTX prophage in the genome of the Mozambique strain was unknown. In this study, pulsed field gel electrophoresis (PFGE) of the whole genome along with Southern hybridization experiments indicated that the classical CTX prophage present in the Mozambique strain is located in the small chromosome. To determine the CTX prophage integration site in the small chromosome of Mozambique strain, the 5′and 3′ junctions of the prophage and small chromosome were PCR amplified, cloned and sequenced. Sequence analysis indicated that the prophage was integrated in the conserved dif site of the replication terminus region of the Mozambique strain. While using an O1 El Tor isolate VC44 as a control strain, which carries tandem copies of CTX prophage in its small chromosome like the Mozambique strain, it was unexpectedly detected that the strain VC44 also possesses classical cholera toxin B gene allele. Since the strain VC44 was isolated in India in the year 1992, it appears that the Mozambique strain has probably originated from a VC44-like strain.
Keywords: Classical CTX prophage; Small chromosome; Vibrio cholerae ; Hybrid; dif/attB ; attP