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Archives of Microbiology (v.178, #4)
No Title by Julia A. Vorholt (pp. 239-249).
Methylotrophic bacteria can grow on a number of substrates as energy source with only one carbon atom, such as methanol, methane, methylamine, and dichloromethane. These compounds are metabolized via the cytotoxin formaldehyde. The formaldehyde consumption pathways, especially the pathways for the oxidation of formaldehyde to CO2 for energy metabolism, are a central and critical part of the metabolism of these aerobic bacteria. Principally, two main types of pathways for the conversion of formaldehyde to CO2 have been described: (1) a cyclic pathway initiated by the condensation of formaldehyde with ribulose monophosphate, and (2) distinct linear pathways that involve a dye-linked formaldehyde dehydrogenase or C1 unit conversion bound to the cofactors tetrahydrofolate (H4F), tetrahydromethanopterin (H4MPT), glutathione (GSH), or mycothiol (MySH). The pathways involving the four cofactors have in common the following sequence of events: the spontaneous or enzyme-catalyzed condensation of formaldehyde and the respective C1 carrier, the oxidation of the cofactor-bound C1 unit and its conversion to formate, and the oxidation of formate to CO2. However, the H4MPT pathway is more complex and involves intermediates that were previously known solely from the energy metabolism of methanogenic archaea. The occurrence of the different formaldehyde oxidation pathways is not uniform among different methylotrophic bacteria. The pathways are in part also used by other organisms to provide C1 units for biosynthetic reactions (e.g., H4F-dependent enzymes) or detoxification of formaldehyde (e.g., GSH-dependent enzymes).
Keywords: Methylotrophic bacteria Methanogenic archaea Methylobacterium Paracoccus Methanol Formaldehyde Tetrahydrofolate Tetrahydromethanopterin Glutathione Mycothiol
No Title by Daniel J. Arp; Luis A. Sayavedra-Soto; Norman G. Hommes (pp. 250-255).
Nitrosomonas europaea uses only NH3, CO2 and mineral salts for growth and as such it is an obligate chemo-lithoautotroph. The oxidation of NH3 is a two-step process catalyzed by ammonia monooxygenase (AMO) and hydroxylamine oxidoreductase (HAO). AMO catalyzes the oxidation of NH3 to NH2OH and HAO catalyzes the oxidation of NH2OH to NO2 –. AMO is a membrane-bound enzyme composed of three subunits. HAO is located in the periplasm and is a homotrimer with each subunit containing eight c-type hemes. The electron flow from HAO is channeled through cytochrome c 554 to cytochrome c m552, where it is partitioned for further utilization. Among the ammonia-oxidizing bacteria, the genes for AMO, these cytochromes, and HAO are present in up to three highly similar copies. Mutants with mutations in the copies of amoCAB and hao in N. europaea have been isolated. All of the amoCAB and hao gene copies are functional. N. europaea was selected by the United States Department of Energy for a whole-genome sequencing project. In this article, we review recent research on the molecular biology and biochemistry of NH3 oxidation in nitrifiers.
Keywords: Nitrosomonas europaea Nitrification Ammonia oxidation Obligate chemo-lithoautotrophy AMO mutant strains HAO mutant strains Bioremediation Cytochromes Electron flow
No Title by Hong Li; Louis A. Sherman (pp. 256-266).
The impact of nitrogen deficiency on the unicellular cyanobacterium Synechocystis sp. strain PCC 6803 and three nbl (non-bleaching) mutants (ΔnblA1, ΔnblA2 and ΔnblB) was investigated. The ΔnblA mutants entered a non-dividing, dormant state soon after the initiation of nitrogen starvation. The cells became larger, the membrane system was disorganized, and ribosomes were found near the membranes much less frequently. Photosystem II (PSII) activity declined to approximately 10% of the wild-type level and the amount of D1 protein declined precipitously, despite adequate psbA transcription; PSI activity declined, but more slowly. Transcription from PSII (except psbA), PSI and phycobilisome genes was very low. Fluorescence at 77K indicated many partially assembled or unassembled phycobilisomes. The level of transcript accumulation increased to normal by 4 h after the readdition of nitrogen to the culture. When NblA was present, the phycobilisomes were degraded to provide a nitrogen source for continued growth and metabolism. An important difference between the wild-type, mutant ΔnblB, and the ΔnblA mutants was seen in the rod linker proteins. Under nitrogen-deprivation condition, the LR33 and LR34.5 linker proteins were extensively degraded in the wild-type and ΔnblB mutant, but remained intact in the ΔnblA mutants.
Keywords: Cyanobacteria Phycobilisomes Nitrogen deficiency Synechocystis sp. strain PCC 6803 Gene expression Photosynthesis Stress response
No Title by Mirko Glinski; Claus Urbanke; Till Hornbogen; Rainer Zocher (pp. 267-273).
Enniatin synthetase (Esyn), a 347-kDa multienzyme consisting of two substrate activation modules, is responsible for the nonribosomal formation of the cyclohexadepsipeptide enniatin. The synthesis follows the so-called thiol template mechanism. While this process is basically well established, no substantial insight into the 3-dimensional arrangement of these enzymes and possible interactions between them exists to date. To find out whether enniatin synthesis is an intramolecular process or the result of three interacting Esyn molecules (intermolecular), analytical ultracentrifugation equilibration studies were carried out. The molecular mass of Esyn was determined by ultracentrifugation and is in good agreement with that calculated from the ORF of the encoding gene, indicating that Esyn exists in solution as a monomer. This strongly suggests that synthesis of the cyclohexadepsipeptide enniatin follows an intramolecular reaction mechanism in which all three reaction cycles are catalyzed by a single Esyn molecule. This finding was supported by in vitro complementation studies in which [14C]-methylvalyl Esyn, upon incubation with the second substrate D-2-hydroxyisovaleric acid (D-Hiv) and ATP, did not yield radioactive enniatin. This confirms our previous assumption of an iterative reaction mechanism similar to that for fatty acid synthase. Furthermore, the sedimentation rate constant evaluated from analytical ultracentrifugation was lower (S20,w=14.1S) than expected (S20,w=16.9S) for a globular protein, indicating that Esyn has an extended structure.
Keywords: Analytical ultracentrifugation Enniatin synthetase Fusarium scirpi N-methylcyclopeptides Nonribosomal peptide synthesis Thiol template mechanism
No Title by Richard O. Jenkins; Sarah N. Forster; Peter J. Craig (pp. 274-278).
Pure cultures of an aerobically grown Flavobacterium sp. were shown by hydride generation–cold trap–atomic absorption spectrometry to biomethylate inorganic antimony (III) supplied as potassium antimony tartrate. Growth inhibition of the Flavobacterium sp. by antimony (III) over the range 0–30 mg Sb l–1 was assessed by optimising parameters within an extended logistic growth model. Antimony (III) concentrations over this range influenced both the extent of antimony biomethylation (up to 4.0 µg l–1) and the relative proportions of the involatile mono-, di, and trimethylantimony species formed. Provision of inorganic arsenic (III) alongside antimony (III) enhanced formation of the involatile methylantimony species up to eight-fold. The data are consistent with accumulation of involatile intermediates from an antimony or arsenic biomethylation pathway in culture supernatants. Low yields of methylantimony species (<0.03%) suggest that antimony biomethylation by the Flavobacterium sp. was a fortuitous rather than a primary resistance mechanism for this element. These findings demonstrate that anaerobiosis is not an obligate requirement for methylantimony formation in prokaryotes, thus broadening the range of habitats for potential formation of methylantimony species in nature.
Keywords: Antimony Biomethylation Bacteria Flavobacterium Trimethylantimony Methylantimony
No Title by Jean-François Rontani; Abdelkrim Mouzdahir; Valérie Michotey; Patricia Bonin (pp. 279-287).
The aerobic and anaerobic metabolism of the isoprenoid alkene squalene was investigated in a new type of marine denitrifying bacterium, strain 2sq31, isolated from marine sediment. Strain 2sq31 was identified as a species of Marinobacter. Under denitrifying conditions, the strain efficiently degraded squalene; of 0.7 mmol added per liter of medium, 77% was degraded within 120 days under anoxic conditions with nitrate as electron acceptor. Tertiary diols and methyl ketones were identified as metabolites, and an anaerobic pathway was suggested to explain the formation of such compounds. The first step in anaerobic degradation of squalene by strain 2sq31 involves hydration of double bonds to tertiary alcohols. Under oxic conditions, the degradation of squalene by strain 2sq31 was rapid and involved oxidative splitting of the C-10/C-11 or C-14/C-15 double bonds, in addition to the pathways observed under denitrifying conditions.
Keywords: Marinobacter sp. strain 2sq31 Squalene Aerobic metabolism Anaerobic metabolism Denitrifying conditions Marine sediments
No Title by Anahita Dastur; Pradeep Kumar; Sneha Ramesh; Mundodi Vasanthakrishna; Umesh Varshney (pp. 288-296).
Initiation of protein synthesis is a major post-transcriptional regulatory step in gene expression. The initiator tRNA gene from Mycobacterium smegmatis, a fast-growing mycobacterium, was characterized and compared with its counterpart from Mycobacterium tuberculosis, a slow-growing mycobacterium. In both mycobacteria, the functional initiator tRNA genes were found in a single copy. Unlike the M. tuberculosis initiator tRNA, the CCA end of the M. smegmatis initiator is not encoded in the gene, and it is most likely added post-transcriptionally. Transcription start site mapping allowed accurate assignment of the hexameric –10 and –35 promoter elements for both genes. These elements of the M. smegmatis initiator tRNA gene contain single nucleotide changes compared to their respective counterparts in the M. tuberculosis gene. Chloramphenicol acetyl transferase reporter assays suggested that the promoter of the initiator tRNA gene from M. smegmatis is twice as strong as that of M. tuberculosis, irrespective of whether the assays were performed in the fast-growing homologous host (M. smegmatis) or the slow-growing heterologous host (M. tuberculosis). Characterization of the M. smegmatis metU promoter, in this study, provides a valuable tool for the expression of genes in mycobacteria.
Keywords: Mycobacterium tuberculosis Mycobacterium smegmatis Protein synthesis metU metA Transcription start site
No Title by Ida Steen; Torleiv Lien; Marit Madsen; Nils-Kåre Birkeland (pp. 297-300).
The role of Asp-328 and Ile-329 as a cofactor discrimination site of the NAD-dependent isocitrate dehydrognase (NAD-IDH) from Pyrococcus furiosus has been verified by replacing these residues with Lys and Tyr, respectively, which are the corresponding residues in NADP-IDH from Escherichia coli. The Asp-328–Lys mutant showed dual coenzyme specificity, whereas introduction of the double mutation, Asp-328–Lys/Ile-329–Tyr shifted the cofactor preference from NAD to NADP. NADP-dependent P. furiosus IDH retained thermostability and thermoactivity compared with NAD-IDH.
Keywords: Pyrococcus furiosus Hyperthermophile Isocitrate dehydrogenase idh Isopropylmalate dehydrogenase Cofactor specificity
No Title by En Wang; Antonio M. Rogel; Xin Sui; Wen Chen; Esperanza Martínez-Romero; Peter van Berkum (pp. 301-305).
Amorpha fruticosa was inoculated with rhizosphere soil from Iowa, USA, and 140 rhizobia isolated from root nodules were compared with Mesorhizobium amorphae originating from Chinese soils. PCR-RFLP patterns of the 16S rRNA gene from the isolates and from M. amorphae were the same. All isolates had a symbiotic plasmid of the same size with a single nifH gene. DNA:DNA hybridization values, DNA G+C content, and induced Nod factor patterns also were similar. We concluded that the four genotypes distinguished among 53 representative American isolates were M. amorphae. Since A. fruticosa is native to the Americas and is highly specific in its nodulation requirement, M. amorphae probably was transmitted to China.
Keywords: Amorpha fruticosa Mesorhizobium amorphae Rhizobia Symbiosis Nitrogen fixation Plant-microbe interaction
No Title by Sharyn L. Rusch; Cynthia L. Mascolo; Maha O. Kebir; Debra A. Kendall (pp. 306-310).
In Escherichia coli, exported proteins are synthesized as precursors containing an amino-terminal signal peptide which directs transport through the translocase to the proper destination. We have constructed a series of signal peptide mutants, incorporating linker sequences of varying lengths between the amino-terminal charge and core region hydrophobicity, to examine the requirement for the juxtaposition of these two structural features in promoting protein transport. In vivo and in vitro analyses indicated that high transport efficiency via signal peptides with core regions of marginal hydrophobicity absolutely requires the proximity of sufficient charge.
Keywords: Hydrophobicity Protein transport SecA Signal peptide
No Title by Rebecca Archuleta; Patricia Mullens; Todd P. Primm (pp. 311-314).
Mycobacterium avium grew in media at 14–37 °C, and persisted at 4 °C and 42 °C. The bacteria lost approximately 90% viability after 3 months in reverse-osmosis deionized water at 4–37 °C. Cooler temperatures lowered the death rate. Death rates also decreased after a 5- to 10-day starvation adaptation period. Alterations of the steady-state levels of different mycolic acid classes, presumably to facilitate thermoadaptation, were found. Following desiccation, M. avium lost viability at a constant rate (half-life of 2.3 days). This implies that bacilli contaminating dry medical surfaces would persist for short periods of time. The remarkable stress survival exhibited by M. avium further suggests persistence in a range of environmental and clinical settings.
Keywords: Starvation Mycobacterium avium Desiccation Stress survival Mycolic acids