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Archives of Microbiology (v.182, #5)
Random transposon mutagenesis of Lactobacillus plantarum by using the pGh9:ISS1 vector to clone genes involved in the regulation of phenolic acid metabolism by Jérôme Gury; Lise Barthelmebs; Jean-François Cavin (pp. 337-345).
The lactic acid bacterium Lactobacillus plantarum possesses a promising inducible padA promoter that controls the expression of the padA gene encoding a phenolic acid decarboxylase, and which is transcriptionally regulated by phenolic acids. A strategy was followed in order to clone genes involved in the transcriptional regulation of the padA gene. The pGh9:ISS1 plasmid was used to perfect the mutagenesis of L. plantarum by transposition. This plasmid transposed randomly in the L. plantarum NC8 chromosome, with a frequency of 0.03% at a non-permissive replicating temperature of 42°C. A L. plantarum mutant strain harbouring the transcriptional fusion padA∷cat that displayed a chloramphenicol acetyl transferase (CAT) activity induced by p-coumaric acid was constructed. This strain was mutated by transposition with pGh9:ISS1 in order to select mutants with a constitutive CAT activity therefore able to grow on medium containing chloramphenicol without the phenolic acid inducer. Four mutants were identified and three of them harboured an inserted single copy of the pGh9:ISS1 vector. Analysis of ISS1 target sites allowed the identification of two genes: one encoding a putative protein that displays similarity with histidine-binding protein, the other belongs to the ATP-dependant RNA helicase family, protein which are usually involved in gene expression.
Keywords: Lactobacillus plantarumMutagenesis; TranspositionpadA gene regulation
Isogenic auxotrophic mutant strains in the Aspergillus fumigatus genome reference strain AF293 by Tao Xue; Cuong K. Nguyen; Angela Romans; Dimitrios P. Kontoyiannis; Gregory S. May (pp. 346-353).
Aspergillus fumigatus is a ubiquitous fungus that is a frequent opportunistic pathogen in immunosuppressed patients. Because of its role as a pathogen, it is of considerable experimental interest. A set of auxotrophic isogenic strains in the A. fumigatus genome reference strain AF293 has been developed. Using molecular genetic methods, arginine and lysine auxotrophs were made by deletion of argB and lysB, respectively. Transformation of these auxotrophic strains with plasmids carrying argB or lysB, respectively, results in efficient integration at these loci. Finally, these strains are able to form stable diploids, which should further facilitate analysis of gene functions in this fungus. Furthermore, the development of this isogenic set of auxotrophic strains in the AF293 background will enable investigators to study this important opportunistic human pathogen with greater facility.
Keywords: Auxotroph; Transformation; Homologous recombination
Roles of pyruvate kinase and malic enzyme in Corynebacterium glutamicum for growth on carbon sources requiring gluconeogenesis by Roman Netzer; Malgorzata Krause; Doris Rittmann; Petra G. Peters-Wendisch; Lothar Eggeling; Volker F. Wendisch; Hermann Sahm (pp. 354-363).
In many bacteria, pyruvate kinase serves a well-defined function in glycolysis, catalyzing an ATP-generating reaction. However, its role during growth on carbon sources requiring glucoeneogenesis is less well investigated. We analyzed a defined pyruvate kinase gene (pyk) deletion mutant of Corynebacterium glutamicum, which is unable to grow on ribose as sole carbon source. Unexpectedly, the pyk deletion mutant was also unable to grow on acetate or citrate as sole carbon sources unless low amounts of pyruvate were added to the growth medium. A spontaneous suppressor mutant of the pyk deletion strain that regained the ability to grow on acetate was isolated. DNA microarray experiments revealed increased expression of the malic enzyme gene malE. The point mutation upstream of malE identified in this mutant was responsible for the loss of carbon-source-dependent regulation, as revealed by transcriptional fusion analysis. Overexpression of malE was sufficient to restore growth of the pyk deletion strain on acetate or citrate. The requirement of increased malic enzyme levels to re-route the carbon flux at the interface between glycolysis, gluconeogenesis and the tricarboxylic acid cycle in order to compensate for the absence of pyruvate kinase indicates a metabolic flux bifurcation at the metabolic node phosphoenolpyruvate. Whereas during growth of C. glutamicum on acetate or citrate most of the phosphoenolpyruvate generated from oxaloacetate is metabolized in gluconeogenesis, a fraction is converted by pyruvate kinase in the glycolytic direction to sustain proper pyruvate availability for biomass synthesis.
Characterisation of oxidative stress-resistant mutants of Streptococcus thermophilus CNRZ368 by Annabelle Fernandez; Annabelle Thibessard; Frédéric Borges; Brigitte Gintz; Bernard Decaris; Nathalie Leblond–Bourget (pp. 364-372).
During industrial processes, the dairy organism Streptococcus thermophilus is exposed to stress conditions. Its ability to survive and grow in an aerobic environment indicates that it must possess defensive mechanisms against reactive oxygen species. To identify the genes involved in oxidative stress defence, a collection of mutants was generated by random insertional mutagenesis and screened for menadione sensitivity and resistance. Results obtained for resistant clones allowed the identification of eight loci. The insertions affected genes whose homologues in other bacteria were previously identified as being involved in stress response (deoB, gst) or transcription regulation (rggC) and five ORFs of unknown function. The tolerance of the eight mutants to air-exposure, methyl viologen and H2O2 was studied. Real-time quantitative PCR was used to analyse the transcript level of mutated genes and revealed that most were down-regulated during oxidative stress.
Keywords: Streptococcus thermophilusOxidative stress; Real-time quantitative RT-PCR
Chemolithoautotrophy in the marine, magnetotactic bacterial strains MV-1 and MV-2 by Dennis A. Bazylinski; Annette J. Dean; Timothy J. Williams; Linda Kimble Long; Shawna L. Middleton; Bradley L. Dubbels (pp. 373-387).
Magnetite-producing magnetotactic bacteria collected from the oxic–anoxic transition zone of chemically stratified marine environments characterized by O2/H2S inverse double gradients, contained internal S-rich inclusions resembling elemental S globules, suggesting they oxidize reduced S compounds that could support autotrophy. Two strains of marine magnetotactic bacteria, MV-1 and MV-2, isolated from such sites grew in O2-gradient media with H2S or thiosulfate (S2O32−) as electron sources and O2 as electron acceptor or anaerobically with S2O32− and N2O as electron acceptor, with bicarbonate (HCO3−)/CO2 as sole C source. Cells grown with H2S contained S-rich inclusions. Cells oxidized S2O32− to sulfate (SO42−). Both strains grew microaerobically with formate. Neither grew microaerobically with tetrathionate (S4O62−), methanol, or Fe2+ as FeS, or siderite (FeCO3). Growth with S2O32− and radiolabeled 14C-HCO3− showed that cell C was derived from HCO3−/CO2. Cell-free extracts showed ribulose 1,5-bisphosphate carboxylase/oxygenase (RubisCO) activity. Southern blot analyses indicated the presence of a form II RubisCO (cbbM) but no form I (cbbL) in both strains. cbbM and cbbQ, a putative post-translational activator of RubisCO, were identified in MV-1. MV-1 and MV-2 are thus chemolithoautotrophs that use the Calvin–Benson–Bassham pathway. cbbM was also identified in Magnetospirillum magnetotacticum. Thus, magnetotactic bacteria at the oxic–anoxic transition zone of chemically stratified aquatic environments are important in C cycling and primary productivity.
Keywords: Calvin–Benson–Bassham pathwaycbbMcbbQChemolithoautotrophy; Chemoorganoautotrophy; Magnetite; Magnetotactic bacterium; Reductive pentose phosphate pathway; Ribulose 1,5-bisphosphate carboxylase/oxygenase
Characterization of the polyketide spore pigment cluster whiESa in Streptomyces aureofaciens CCM3239 by Renata Novakova; Jana Bistakova; Jan Kormanec (pp. 388-395).
A spore pigment polyketide gene cluster, whiESa, was cloned from Streptomyces aureofaciens CCM3239 using a probe from the S. coelicolor A3(2) whiE gene cluster. Sequence analysis of a 4,657-bp DNA fragment revealed five open reading frames with the highest similarity to the S. coelicolor A3(2) whiE locus responsible for spore pigment biosynthesis, with conservation of the size and position of the genes. The whiESa gene cluster was disrupted by a homologous recombination in S. aureofaciens CCM3239, replacing the most important whiESaIII gene encoding ketosynthase with a thiostrepton resistance gene. The mutation affected spore pigmentation. In contrast to wild-type grey-pink spore pigmentation, the mutant produced white spores, although overall spore morphology was not affected. Transcriptional analysis of whiESa revealed two divergently oriented promoters, whiESap1 and whiESap2, upstream of the whiESaI and whiESaVIII genes, respectively. Both promoters were developmentally regulated in S. aureofaciens CCM3239. They were induced at the late stages of differentiation, during sporulation of aerial hyphae and were dependent upon early sporulation-specific sigma factor σRpoZ and putative transcription factor WhiB. The level of the transcript originating from the whiESap2 promoter was substantially reduced in a sigF mutant of S. aureofaciens CCM3239, indicating its dependence upon the late sporulation sigma factor σF. Comparison of the whiE promoters in three different spore pigment polyketide clusters revealed a highly conserved region upstream of the −35 promoter region that may bind a transcriptional regulator.
Keywords: Differentiation; Gene disruption; Promoter; Sigma factorStreptomyces aureofaciens
d-Amino acid-N-acetyltransferase of Saccharomyces cerevisiae: a close homologue of histone acetyltransferase Hpa2p acting exclusively on free d-amino acids by Geok-Yong Yow; Takuma Uo; Tohru Yoshimura; Nobuyoshi Esaki (pp. 396-403).
d-Amino acid N-acetyltransferase is a unique enzyme of Saccharomyces cerevisiae acting specifically on d-amino acids. The enzyme was found to be encoded by HPA3, a putative histone/protein acetyltransferase gene, and we purified its gene product, Hpa3p, from recombinant Escherichia coli cells. Hpa3p shares 49% sequence identity and 81% sequence similarity with a histone acetyltransferase, Hpa2p, of S. cerevisiae. Hpa3p acts on a wide range of d-amino acids but shows extremely low activity toward histone. However, Hpa2p does not act on any of the free amino acids except l-lysine and d-lysine. Kinetic analyses suggest that Hpa3p catalyzes the N-acetylation of d-amino acids through an ordered bi-bi mechanism, in which acetyl-CoA is the first substrate to be bound and CoA is the last product to be liberated.
Keywords: d-Amino acidd-Amino acid N-acetyltransferaseHPA3Saccharomyces cerevisiaeYeast
Composition of the lipids of Nanoarchaeum equitans and their origin from its host Ignicoccus sp. strain KIN4/I by Ulrike Jahn; Roger Summons; Helen Sturt; Emmanuelle Grosjean; Harald Huber (pp. 404-413).
The contents and nature of the membrane lipids of Nanoarchaeum equitans and Ignicoccus sp. strain KIN4/I, grown at 90°C, and Ignicoccus sp. strain KIN4/I, cultivated at its lowest and highest growth temperatures (75°C and 95°C) were analyzed. Both organisms contained very simple and qualitatively identical assemblages of glycerol ether lipids, showing only differences in the amounts of certain components. LC–MS analyses of the total lipid extracts revealed that archaeol and caldarchaeol were the main core lipids. The predominant polar headgroups consisted of one or more sugar residues attached either directly to the core lipid or via a phosphate group. GC–MS analyses of hydrolyzed total lipid extracts revealed that the co-culture of N. equitans and Ignicoccus sp. strain KIN4/I, as well as Ignicoccus sp. strain KIN4/I grown at 90°C, contained phytane and biphytane in a ratio of approximately 4:1. Purified N. equitans cells and Ignicoccus sp. strain KIN4/I cultivated at 75°C and 95°C had a phytane to biphytane ratio of 10:1. Sugar residues were mainly mannose and small amounts of glucose. Consistent 13C fractionation patterns of isoprenoid chains of N. equitans and its host indicated that the N. equitans lipids were synthesized in the host cells.
Keywords: Nanoarchaeum equitansIgnicoccusLipid analysis; Intact polar lipids; Archaeol; Caldarchaeol; Glycerol dibiphytanyl glycerol tetraether
Oxaloacetate decarboxylase of Archaeoglobus fulgidus: cloning of genes and expression in Escherichia coli by Pius Dahinden; Klaas M. Pos; Malgorzata Taralczak; Peter Dimroth (pp. 414-420).
Archaeoglobus fulgidus harbors three consecutive and one distantly located gene with similarity to the oxaloacetate decarboxylase Na+ pump of Klebsiella pneumoniae (KpOadGAB). The water-soluble carboxyltransferase (AfOadA) and the biotin protein (AfOadC) were readily synthesized in Escherichia coli, but the membrane-bound subunits AfOadB and AfOadG were not. AfOadA was affinity purified from inclusion bodies after refolding and AfOadC was affinity purified from the cytosol. Isolated AfOadA catalyzed the carboxyltransfer from [4-14C]-oxaloacetate to the prosthetic biotin group of AfOadC or the corresponding biotin domain of KpOadA. Conversely, the carboxyltransferase domain of KpOadA exhibited catalytic activity not only with its pertinent biotin domain but also with AfOadC.
Keywords: Archaeoglobus fulgidusOxaloacetate decarboxylase; Na+ pump; Archaeal membrane protein; Protein targeting and translocation; Refolding of inclusion bodies
A naturally occurring point mutation in the 13-mer R repeat affects the oriC function of the large chromosome of Vibrio cholerae O1 classical biotype by Arjun Saha; Shruti Haralalka; Rupak K. Bhadra (pp. 421-427).
The genome of Vibrio cholerae consists of two circular chromosomes of different sizes. Here, a comparative analysis of the replication origins of the large chromosomes (oriCIVC) of classical and El Tor biotypes of the pathogen is reported. Extensive nucleotide sequence analyses revealed that the oriCIVC region has six DnaA boxes instead of the five found in Escherichia coli oriC. The additional DnaA box, designated RV, was unique in V. cholerae as well as in other members of the family Vibrionaceae. However, RV was not found to be essential for the autonomous replication function of the 307-bp oriCIVC minimal region. In contrast to El Tor and the recently evolved V. cholerae O139 strains, the oriCIVC region of the classical biotype showed only a single base transition (T→G) in a highly conserved AT-rich 13-mer R repeat region. From the minichromosome copy number and its transformational efficiency analyses, it appears that the single base substitution in the oriCIVC of the classical biotype has a significant effect on its replication initiation.
Keywords: Vibrio choleraeLarge chromosomeoriCDnaA box; 13-mer repeat