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Archives of Microbiology (v.190, #1)
Involvement of GlnK, a PII protein, in control of nitrogen fixation and ammonia assimilation in Pseudomonas stutzeri A1501 by Sheng He; Ming Chen; Zhihong Xie; Yongliang Yan; Hongquan Li; Ying Fan; Shuzhen Ping; Min Lin; Claudine Elmerich (pp. 1-10).
The nitrogen-fixing, root-associated strain Pseudomonas stutzeri A1501 carries a single gene encoding a protein from the PII family, designated glnK. The glnK gene is co-transcribed with two distantly related copies of amtB genes encoding putative ammonium channels. Transcription of glnK was decreased in the presence of ammonia and was partly dependent on NtrC and RpoN under nitrogen-limiting conditions. Inactivation of glnK led to a mutant strain devoid of nitrogenase activity, auxotrophic for glutamine and unable to deadenylylate glutamine synthetase, while inactivation of amtB 1 led to a prototrophic and Nif+ mutant strain. RT-PCR analysis showed that nifA transcription was abolished in the glnK mutant, while glnA remained transcribed. Using the yeast two-hybrid system, an interaction between GlnK and the C-terminal domain of NifL was observed, suggesting GlnK-dependent control of NifA activity by NifL. Introduction of a plasmid that expressed nifA from a constitutive promoter restored nitrogen fixation to the glnK mutant, and nitrogenase activity was observed even in the presence of ammonia. GlnK signalling appears to be a key regulatory element in control of ammonia assimilation, of nifA expression and in modulation of NifA activity by NifL.
Keywords: Pseudomonas stutzeri ; Nitrogen fixation; Ammonia assimilation; Glutamine synthetase; GlnK; PII ; nif gene regulation; Yeast-two hybrid assay
Different bacterial strategies to degrade taurocholate by Verena Rösch; Karin Denger; David Schleheck; Theo H. M. Smits; Alasdair M. Cook (pp. 11-18).
Aerobic enrichment cultures with taurocholate or alkanesulfonates as sole sources of carbon and energy for growth were successful and yielded nine bacterial isolates, all of which utilized taurocholate. Growth was complex and involved not only many, usually transient, excretion products but also sorption of taurocholate and cholate to cells. Three metabolic strategies to dissimilate taurocholate were elucidated, all of which involved bile salt hydrolase cleaving taurocholate to cholate and taurine. Comamonas testosteroni KF-1 utilized both the taurine and the cholate moieties for growth. Pseudomonas spp., e.g. strain TAC-K3 and Rhodococcus equi TAC-A1 grew with the cholate moiety and released taurine quantitatively. Delftia acidovorans SPH-1 utilized the taurine moiety and released cholate.
Keywords: Bile salt hydrolase; Comamonas testosteroni ; Delftia acidovorans ; Desulfonation; Excretion of cholate; Excretion of taurine; Pseudomonas spp.; Taurine dissimilation
Phylogenetic diversity of nitrogen-fixing bacteria in mangrove sediments assessed by PCR–denaturing gradient gel electrophoresis by Yanying Zhang; Junde Dong; Zhihao Yang; Si Zhang; Youshao Wang (pp. 19-28).
Culture-independent PCR–denaturing gradient gel electrophoresis (DGGE) was employed to assess the composition of diazotroph species from the sediments of three mangrove ecosystem sites in Sanya, Hainan Island, China. A strategy of removing humic acids prior to DNA extraction was conducted, then total community DNA was extracted using the soil DNA kit successfully for nifH PCR amplification, which simplified the current procedure and resulted in good DGGE profiles. The results revealed a novel nitrogen-fixing bacterial profile and fundamental diazotrophic biodiversity in mangrove sediments, as reflected by the numerous bands present DGGE patterns. Canonical correspondence analysis (CCA) revealed that the sediments organic carbon concentration and available soil potassium accounted for a significant amount of the variability in the nitrogen-fixing bacterial community composition. The predominant DGGE bands were sequenced, yielding 31 different nifH sequences, which were used in phylogenetic reconstructions. Most sequences were from Proteobacteria, e.g. α, γ, β, δ-subdivisions, and characterized by sequences of members of genera Azotobacter, Desulfuromonas, Sphingomonas, Geobacter, Pseudomonas, Bradyrhizobium and Derxia. These results significantly expand our knowledge of the nitrogen-fixing bacterial diversity of the mangrove environment.
Keywords: Mangrove sediments; Nitrogen-fixing bacteria; Denaturing gradient gel electrophoresis; Multivariate analysis
Pseudomonas pseudoalcaligenes KF707 upon biofilm formation on a polystyrene surface acquire a strong antibiotic resistance with minor changes in their tolerance to metal cations and metalloid oxyanions by Valentina Tremaroli; Stefano Fedi; Raymond J. Turner; Howard Ceri; Davide Zannoni (pp. 29-39).
The susceptibility to various biocides was examined in planktonic cells and biofilms of the obligate aerobe, PCBs degrader, Pseudomonas pseudoalcaligenes KF707. The toxicity of two antibiotics, amikacin and rifampicin, three metalloid oxyanions (AsO2 −, SeO3 2−, TeO3 2−) and three metal cations (Cd2+, Ni2+, Al3+) was tested at two stages of the biofilm-development (4 and 24 h) and compared to planktonic cells susceptibility. Mature biofilms formed in rich (LB, Luria–Bertani) medium were thicker (23 μm) than biofilms grown in minimal (SA saccarose-arginine) medium (13 μm). Early grown (4 h) SA-biofilms, which consisted of a few sparse/attached cells, were 50–100 times more resistant to antibiotics than planktonic cells. Conversely, minor changes in tolerance to metal(loid)s were seen in both SA- and LB-grown biofilms. In contrast to planktonic cells, no reduction of TeO3 2− to elemental Te(0) or SeO3 2− to elemental Se(0) was seen in KF707 biofilms. The data indicate that: (a) metal tolerance in KF707 biofilms, under the growth and exposure conditions described here, is different than antibiotic tolerance; (b) KF707 planktonic cells and biofilms, are almost equally susceptible to killing by metal cations and oxyanions, and (c) biofilm-tolerance to TeO3 2− and SeO3 2− is not linked to metalloid reduction; this means that KF707 planktonic cells and biofilms differ in their physiology and strategy to counteract metalloid toxicity.
Keywords: Pseudomonas pseudoalcaligenes KF707; Biofilm formation; Metal and metalloid tolerance; Polystyrene surface; Calgary device biofilm
Activation of glucose transport under oxidative stress in Escherichia coli by W. Rungrassamee; X. Liu; P. J. Pomposiello (pp. 41-49).
Global transcription studies have identified a large number of redox-responsive genes, although the biological relevance of this regulation has not been experimentally tested. In particular, several genes coding for enzymes involved in glucose metabolism have been identified as redox-responsive in Escherichia coli. However, only zwf, which codes for glucose-6-phosphate dehydrogenase, has been shown experimentally to affect the cellular resistance to oxidative stress. We addressed the question of whether ptsG, coding for the membrane component of the glucose-specific transporter system, and pgi, coding for phosphoglucose isomerase, two additional genes identified in whole-genome functional screens, are indeed relevant in antioxidant defense. PTS assays showed that glucose transport was induced under oxidative stress elicited by the superoxide-producing agent paraquat (PQ). This induction of glucose transport under oxidative stress was dependent on the soxRS genes, coding for a sensor- transcriptional activator system, and ptsG. The binding of purified SoxS to the ptsG promoter region was shown by gel mobility-shift assay, and the activation of the ptsG promoter P1 was demonstrated by primer extension assays. Finally, a ptsG mutant strain was hypersensitive to PQ when grown in rich medium plus glucose, but not in rich medium without glucose. The pgi gene showed the same pattern of regulation by oxidative stress under the control of the SoxRS system, and a strain carrying a pgi deletion was hypersensitive to PQ.
Ladderane lipid distribution in four genera of anammox bacteria by Jayne E. Rattray; Jack van de Vossenberg; Ellen C. Hopmans; Boran Kartal; Laura van Niftrik; W. Irene C. Rijpstra; Marc Strous; Mike S. M. Jetten; Stefan Schouten; Jaap S. Sinninghe Damsté (pp. 51-66).
Intact ladderane phospholipids and core lipids were studied in four species of anaerobic ammonium oxidizing (anammox) bacteria, each representing one of the four known genera. Each species of anammox bacteria contained C18 and C20 ladderane fatty acids with either 3 or 5 linearly condensed cyclobutane rings and a ladderane monoether containing a C20 alkyl moiety with 3 cyclobutane rings. The presence of ladderane lipids in all four anammox species is consistent with their putative physiological role to provide a dense membrane around the anammoxosome, the postulated site of anammox catabolism. In contrast to the core lipids, large variations were observed in the distribution of ladderane phospholipids, i.e. different combinations of hydrophobic tail (ladderane, straight chain and methyl branched fatty acid) types attached to the glycerol backbone sn-1 position, in combination with different types of polar headgroup (phosphocholine, phosphoethanolamine or phosphoglycerol) attached to the sn-3 position. Intact ladderane lipids made up a high percentage of the lipid content in the cells of “Candidatus Kuenenia stuttgartiensis”, suggesting that ladderane lipids are also present in membranes other than the anammoxosome. Finally, all four investigated species contained a C27 hopanoid ketone and bacteriohopanetetrol, which, indicates that hopanoids are anaerobically synthesised by anammox bacteria.
Keywords: Anammox bacteria; Ladderane lipids; Fatty acids; Hopanoids; High-performance liquid chromatography; Gas chromatography/mass spectrometry
Introduction of a novel pathway for IAA biosynthesis to rhizobia alters vetch root nodule development by Serena Camerini; Beatrice Senatore; Enza Lonardo; Esther Imperlini; Carmen Bianco; Giancarlo Moschetti; Giuseppe L. Rotino; Bruno Campion; Roberto Defez (pp. 67-77).
We introduced into Rhizobium leguminosarum bv. viciae LPR1105 a new pathway for the biosynthesis of the auxin, indole-3-acetic acid (IAA), under the control of a stationary phase-activated promoter active both in free-living bacteria and bacteroids. The newly introduced genes are the iaaM gene from Pseudomonas savastanoi and the tms2 gene from Agrobacterium tumefaciens. Free-living bacteria harbouring the promoter-iaaMtms2 construct release into the growth medium 14-fold more IAA than the wild-type parental strain. This IAA overproducing R. l. viciae, the RD20 strain, elicits the development of vetch root nodules containing up to 60-fold more IAA than nodules infected by the wild-type strain LPR1105. Vetch root nodules derived from RD20 are fewer in number per plant, heavier in terms of dry weight and show an enlarged and more active meristem. A significant increase in acetylene reduction activity was measured in nodules elicited in vetch by RD20.
Keywords: IAA; ARA; Auxin; Nitrogen fixation; Nodule zone; Vicia hirsuta
Two highly thermostable paralogous single-stranded DNA-binding proteins from Thermoanaerobacter tengcongensis by Marcin Olszewski; Małgorzata Mickiewicz; Józef Kur (pp. 79-87).
The thermophilic bacterium Thermoanaerobacter tengcongensis has two single-stranded DNA-binding (SSB) proteins, designated TteSSB2 and TteSSB3. In a SSB complementation assay in Escherichia coli, only TteSSB3 took over the in vivo function of EcoSSB. We have cloned the ssb genes obtained by PCR and have developed E. coli overexpression systems. The TteSSB2 and TteSSB3 consist of 153 and 150 amino acids with a calculated molecular mass of 17.29 and 16.96 kDa, respectively. They are the smallest known bacterial SSB proteins. The homology between amino acid sequences of these proteins is 40% identity and 53% similarity. They are functional as homotetramers, with each monomer encoding one single-stranded DNA binding domain (OB-fold). In fluorescence titrations with poly(dT), both proteins bind single-stranded DNA with a binding site size of about 40 nt per homotetramer. Thermostability with half-life of about 30 s at 95°C makes TteSSB3 similar to the known SSB of Thermus aquaticus (TaqSSB). The TteSSB2 was fully active even after 6 h incubation at 100°C. Here, we show for the first time paralogous thermostable homotetrameric SSBs, which could be an attractive alternative for known homodimeric thermostable SSB proteins in their applications for molecular biology methods and analytical purposes.
Keywords: SSB; Expression; Purification; Replication; Thermophilic bacteria
Genetic analysis of phenylacetic acid catabolism in Arthrobacter oxydans CECT386 by Juana María Navarro-Llorens; Oliver Drzyzga; Julián Perera (pp. 89-100).
Arthrobacter oxydans CECT386 is a Gram-positive bacterium able to use either phenylacetic acid or phenylacetaldehyde as the sole carbon and energy source for aerobic growth. Genes responsible for the catabolism of these compounds have been located at two chromosomal regions and were organized in one isolated paaN gene and two putative paa operons, one consisting of the paaD, paaF, tetR and prot genes, and one consisting of the paaG, paaH, paaI, paaJ, paaK and paaB genes. The identity of the paaF and paaN genes was supported by functional complementation experiments. A comparison with the paa catabolic genes and/or gene clusters of other bacteria that degrade these aromatic compounds is presented. The results of this study broaden the knowledge regarding the range of metabolic potential of this strain and eventually make it attractive for environmental applications.
Keywords: Phenylacetic acid; Biodegradation; Arthrobacter
Listeria monocytogenes internalins bind to the human intestinal mucin MUC2 by Sara K. Lindén; Hélène Bierne; Christophe Sabet; Chin Wen Png; Timothy H Florin; Michael A. McGuckin; Pascale Cossart (pp. 101-104).
Listeria monocytogenes cross the intestinal barrier causing systemic infections with high mortality rates. Intestinal infection triggers release of intestinal mucus. We show that three L. monocytogenes internalins, InlB, InlC and InlJ all bound to MUC2 (the major component of intestinal mucus), but not to the cell surface mucin MUC1. Binding was strongest to InlB>InlC>InlJ (P < 0.001). Listerial internalins are characterized by their internalin domain, composed by leucine rich repeats (LRR) followed by an immunogloblin-like region. We report here that the internalin domain of the InlJ protein also bound MUC2, suggesting that an internalin domain is sufficient to bind to MUC2.
Keywords: Listeria ; LRR; Mucin; Mucus; MUC2; MUC1
An ABC transporter encoding gene lndW confers resistance to landomycin E by Iryna Ostash; Yuriy Rebets; Bohdan Ostash; Anton Kobylyanskyy; Maksym Myronovskyy; Tatsunosuke Nakamura; Suzanne Walker; Victor Fedorenko (pp. 105-109).
Streptomyces globisporus 1912 produces a polyketide antibiotic landomycin E (LaE), which possesses anticancer activity. A 1.8 kb DNA fragment at the end of landomycin E biosynthetic gene cluster was sequenced. DNA sequence analysis of this fragment identified one complete open reading frame, designated lndW. The deduced sequence of lndW gene product revealed significant similarity to the ATP-binding domains of the ABC (ATP-binding protein cassette) superfamily of transport-related proteins. Knockout of lndW had no significant effect on resistance to LaE and its production. The expression of lndW in S. globisporus 1912 was proven via transcriptional fusion of lndW promoter to EGFP (enhanced green fluorescent protein). Overexpression of lndW in S. lividans TK24 conferred resistance to LaE. The mechanism of lndW function in LaE biosynthesis is discussed.
Keywords: Streptomyces globisporus 1912; Landomycin resistance; ABC transporters