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Archives of Microbiology (v.186, #3)
The rggC locus, with a frameshift mutation, is involved in oxidative stress response by Streptococcus thermophilus by Annabelle Fernandez; Frédéric Borges; Brigitte Gintz; Bernard Decaris; Nathalie Leblond-Bourget (pp. 161-169).
In Streptococcus thermophilus, the locus rggC contains a frameshift mutation and thus consists of two open reading frames (ORFs), rggC 1 and rggC 2, which encode proteins exhibiting similarity with the Rgg transcriptional regulator family. In this work, mutants showing a partial deletion of rggC 1 and rggC 2 were constructed and their response to menadione, a superoxide-generating compound, was analysed. These mutants exhibited different behaviour to this oxidative stress compared with the wild-type strain. Analysis of this locus among 21 strains of S. thermophilus showed a polythymine tract length variability and a strain-dependant adenine residue could be found upstream of this repeat. This interstrain polymorphism supports evidence for the hypothesis that the rggC locus is phase variable.
Keywords: Streptococcus thermophilus ; Oxidative stress response; Transcriptional regulator; Rgg; Genetic variability; Frameshift mutation
Characterization of the Vibrio cholerae vexAB and vexCD efflux systems by James E. Bina; Daniele Provenzano; Chunmei Wang; Xiaowen R. Bina; John J. Mekalanos (pp. 171-181).
Vibrio cholerae is an important human pathogen that causes the diarrheal disease cholera. Colonization of the human host is dependent upon coordinated expression of several virulence factors in response to as yet unknown environmental cues. Bile acids have been implicated in the in vitro regulation of several V. cholerae genes, including those involved in motility, chemotaxis, outer membrane protein production, and virulence factor production. Bile is toxic to bacteria and colonization of the intestinal tract is dependent upon bacterial resistance to bile acids. We have identified and characterized two bile-regulated RND-family efflux systems, named here vexAB and vexCD, that are involved in V. cholerae bile resistance. Mutational analysis revealed that the vexAB system is responsible for in vitro intrinsic resistance of V. cholerae to multiple antimicrobial compounds, including bile acids. In contrast, the vexCD efflux system was specific for certain bile acids and detergents and functioned in conjunction with the vexAB system to provide V. cholerae with high-level bile resistance. Mutants containing deletion of vexB, vexD, and vexB–vexD were able to efficiently colonize the infant mouse suggesting that these efflux systems were dispensable for V. cholerae growth in the small intestines of infant mice.
Keywords: Cholera; Efflux; Bile; RND
Rhodococcus sp. strain TM1 plays a synergistic role in the degradation of piperidine by Mycobacterium sp. strain THO100 by Yong-Hak Kim; Un-Beom Kang; Kyoko Konishi; Cheolju Lee (pp. 183-193).
Mycobacterium sp. strain THO100 and Rhodococcus sp. strain TM1 were isolated from a morpholine-containing enrichment culture of activated sewage sludge. Strain THO100, but not strain TM1, was able to degrade alicyclic amines such as morpholine, piperidine, and pyrrolidine. The mixed strains THO100 and TM1 showed a better growth on piperidine as the substrate than the pure strain THO100 because strain TM1 was able to reduce the level of glutaraldehyde (GA) produced during piperidine degradation. GA was toxic to strain THO100 (IC50 = 28.3 μM) but less toxic to strain TM1 (IC50 = 215 μM). Strain THO100 possessed constitutive semialdehyde dehydrogenases, namely Sad1 and Sad2, whose activities toward succinic semialdehyde (SSA) were strongly inhibited by GA. The two isozymes were identified as catalase–peroxidase (KatG = Sad1) and semialdehyde dehydrogenase (Sad2) based on mass spectrometric analyses of tryptic peptides and database searches of the partial DNA sequences of their genes. In contrast, strain TM1 containing another constitutive enzyme Gad1 could oxidize both SSA and GA. This study suggested that strain TM1 possessing Gad1 played a synergistic role in reducing the toxic and inhibitory effects of GA produced in the degradation of piperidine by strain THO100.
Keywords: Glutaraldehyde; Mycobacterium ; Piperidine; Rhodococcus ; Semialdehyde dehydrogenase
Ultrastructural and chemotaxonomic analysis of a xylanolytic strain of Cryptococcus adeliensis isolated from sheep droppings in Spain by Encarna Velázquez; María del Villar; Isabel Grondona; Enrique Monte; Tomás González-Villa (pp. 195-202).
Cryptococcus adeliensis was initially described as a psycrophilic species containing a single strain CBS 8351T isolated from decayed algae in Terre Adelie (Antartida). Later, a second strain of this species was isolated from an immunosuppressed patient affected by leukaemia in Germany and recently several strains from this species have been found in human patients and pigeon droppings of the same country. In this study, we isolated from sheep droppings in Spain a xylanolytic strain named LEVX01 that was phenotypically related to the strain CBS 8351T and showed a 100% similarity in the D1/D2 domain and 5.8S-ITS region sequences with respect to the remaining described strains of C. adeliensis. These findings suggest that this species has a wide geographical distribution and that the animal faeces are a common habitat for C. adeliensis. The chemotaxonomic analyses showed the absence of detectable amounts of xylose in the cell walls of the strains LEVX01 and CBS8351T in contrast to other Cryptococcus species. Interestingly, the ultrastructural study showed the presence of fimbriae in these two strains that could be involved in the attachment to the host cells and, as occurs in Candida albicans, they could also be a pathogenicity factor for the man.
Keywords: Chemotaxonomy; Cryptococcus adeliensis ; Xylanases; Identification; D1/D2 domain; 5.8S-ITS
Preliminary characterization of a thermostable DNA polymerase I from a mesophilic Bacillus sphaericus strain C3-41 by Han Bei; Liu Haizhou; Hu Xiaomin; Yuan Zhiming (pp. 203-209).
A thermostable DNA polymerase I from a mesophilic Bacillus sphaericus strain C3-41 was characterized in this study. The polI was cloned, sequenced and over-expressed in Escherichia coli. The expressed 110 kDa fusion protein of PolI was stable at 70°C for 1 h. Compared with DNA polymerase I of E. coli (TaKaRa), the relative polymerase activity of this PolI was 3.33 ± 0.1 RFU μl−1 at 37°C using fluorescent quantitative analysis. It showed higher polymerase activity than E. coli PolI at higher temperature, with a relative activity of 3.75 ± 0.1 RFU μl−1 at 70°C. The polI sequence analysis and the protein structure prediction indicated that this protein had a high similarly to other PolI from thermophilic micro-organisms. This information is of importance for future study for evolution of the house-keeping gene polI in entomopathogenic bacterium B. sphaericus.
Keywords: Thermostable DNA polymerase I; Bacillus sphaericus ; Mesophile; Mosquitocidal activity
Access to organic and insoluble sources of phosphorus varies among soil Chytridiomycota by David J. Midgley; Peter M. Letcher; Peter A. McGee (pp. 211-217).
The sources of minerals accessed by fungi in the Chytridiomycota (chytrid) in soil are largely unknown. The ability of ten species of soil chytrids to use various sources of phosphorus was examined in vitro. While all grew on orthophosphate, fifty per cent of isolates grew on phytic acid, and one isolate grew on DNA as the sole source of phosphorus. All isolates solubilised and utilised CaHPO4. Most isolates utilised hydroxyapatite when NH 4 + was the nitrogen source. When ammonium was omitted, 50% of isolates solubilised hydroxyapatite. Many soil chytrids may utilise phosphomonoesters as the sole source of phosphorus, and access to DNA appears limited. We suggest that the capacity to use different sources of phosphorus may influence the diversity of chytrids found in Australian soils.
Keywords: Chytrids; Chytridiomycota; Phosphorus; Nutrition; Ecophysiology; Solubilisation
The Bla2 β-lactamase from the live-vaccine strain of Francisella tularensis encodes a functional protein that is only active against penicillin-class β-lactam antibiotics by Xiaowen R. Bina; Chunmei Wang; Mark A. Miller; James E. Bina (pp. 219-228).
Francisella tularensis ssp. tularensis is a category A select agent and the causal organism for the zoonotic disease tularemia. The vast majority of F. tularensis isolates are β-lactamase-positive. β-lactamase production is widely believed to be responsible for the inefficacy of β-lactams in the treatment of tularemia. In this study, we report the cloning and characterization of the two chromosomally encoded F. tularensis ssp. holarctica live-vaccine strain (LVS) β-lactamases. The two LVS β-lactamases were homologous to F. tularensis Schu S4 open reading frames FTT0681c and FTT0611c and have been named bla1 LVS and bla2 LVS , respectively. Recombinant expression in Escherichia coli suggested that bla1 LVS did not encode a functional β-lactamase, whereas bla2 LVS encoded a functional β-lactamase that hydrolyzed penicillins but was inactive against third-generation cephalosporins, including cefprozil. As both LVS and Schu S4 were susceptible to cefprozil, we developed three new shuttle vectors based on selection for the production of the Blashv-2 extended-spectrum β-lactamase with cefprozil. The resulting shuttle vectors were suitable for recombinant gene expression and complementation studies in LVS and Schu S4.
Keywords: Francisella tularensis ; Shuttle vector; β-lactamase; Antibiotic resistance
The acid tolerance response of Bacillus cereus ATCC14579 is dependent on culture pH, growth rate and intracellular pH by Séverine Thomassin; Michel P. Jobin; Philippe Schmitt (pp. 229-239).
The food pathogen Bacillus cereus is likely to encounter acidic environments (i) in food when organic acids are added for preservation purposes, and (ii) during the stomachal transit of aliments. In order to characterise the acid stress response of B. cereus ATCC14579, cells were grown in chemostat at different pH values (pHo from 9.0 to 5.5) and different growth rates (μ from 0.1 to 0.8 h−1), and were submitted to acid shock at pH 4.0. Cells grown at low pHo were adapted to acid media and induced a significant acid tolerance response (ATR). The ATR induced was modulated by both pHo and μ, and the μ effect was more marked at pHo 5.5. Intracellular pH (pHi) was affected by both pHo and μ. At a pHo above 6, the pHi decreased with the decrease of pHo and the increase of μ. At pHo 5.5, pHi was higher compared to pHo 6.0, suggesting that mechanisms of pHi homeostasis were induced. The acid survival of B. cereus required protein neo-synthesis and the capacity of cells to maintain their pHi and ΔpH (pHi - pHo). Haemolysin BL and non-haemolytic enterotoxin production were both influenced by pHo and μ.
Keywords: Bacillus cereus ; Chemostat; Acid tolerance response; Stress; Intracellular pH
Coordinate synthesis of azurin I and copper nitrite reductase in Alcaligenes xylosoxidans during denitrification by Roger L. Harris; Robert R. Eady; S. Samar Hasnain; R. Gary Sawers (pp. 241-249).
The denitrifying bacterium Alcaligenes xylosoxidans synthesises two azurins (Az), which are termed Az I and Az 2. Both function as effective electron donors to copper nitrite reductase (NiR) in vitro. As a first step towards identifying the physiological relevance of these electron transfer proteins in the denitrification process, the gene (azuA) encoding Az I was characterised and its expression with respect to denitrification determined. We show that the azuA gene from A. xylosoxidans is monocistronic and its expression is increased when cells are grown under denitrifying conditions in the presence of nitrate or nitrite. The expression pattern of azuA was similar, though not identical, to that of the monocistronic nirK gene, which encodes copper NiR, and is in accord with both gene products being synthesised when the bacterium denitrifies. Recombinant Az I was exported to the periplasm of the heterologous host Escherichia coli, was synthesised at very high levels (80 mg purified protein per litre) and was fully loaded with copper. Electron donation from reduced recombinant Az to NiR was indistinguishable from the activity determined with the native protein. Taken together, these findings indicate that in A. xylosoxidans azuA expression is coordinated with denitrification and recombinant Az I is processed and matured in the periplasm of E. coli in the same way it is in A. xylosoxidans.
Keywords: Azurin; Electron transfer; Denitrification; Gene regulation; Nitrite reductase