bluuebunny/crossref_metadata_2025 huggingface dataset:
The ParB protein encoded by the RP4 par region is a Ca2+-dependent nuclease linearizing circular DNA substrates
Authors: Grohmann Elisabeth , Stanzer Thomas , Schwab Helmut
Published: June 2009

The parCBA operon, which together with the parDE operon constitutes an efficient stabilization system of the broad-host-range plasmid RP4, encodes a 20 kDa polypeptide (ParB), which exhibits sequence homology to nucleases. The ParB protein was overexpressed by means of an inducible tac-promoter system. Plate assays with herring sperm DNA as substrate provided evidence for nuclease activity. The ParB nuclease shows specificity for circular DNA substrates and linearizes them regardless of the presence in cis of parts of the RP4 partitioning region. The nuclease activity in vitro is stimulated by the presence of Ca2+ ions. EDTA (5 mM) completely inhibits nuclease activity. By restriction analysis of the ParB-linearized products, cleavage of circular DNA substrates taking place preferentially at specific sites was demonstrated. Run-off sequencing and primer extension analysis of ParB-linearized plasmid DNA revealed a specific target for ParB action adjacent to an AT-rich region containing palindromic sequence elements on a pBR322-derived plasmid.
Biological control of Pythium ultimum by Stenotrophomonas maltophilia W81 is mediated by an extracellular proteolytic activity
Authors: Dunne Colum , Crowley Jer J. , Moënne-Loccoz Yvan , Dowling David N. , Bruijn s , O'Gara Fergal
Published: June 2009

Stenotrophomonas maltophilia strain W81, isolated from the rhizosphere of field-grown sugar beet, produced the extracellular enzymes chitinase and protease and inhibited the growth of the phytopathogenic fungus Pythium ultimum in vitro. The role of these lytic enzymes in the interaction between W81 and P. ultimum was investigated using Tn5 insertion mutants of W81 incapable of producing extracellular protease (W81M1), extracellular chitinase (W81M2) or the two enzymes (W81A1). Lytic enzyme activity was restored in W81A1 following introduction of a 15 kb cosmid-borne fragment of W81 genomic DNA. Incubation of P. ultimum in the presence of commercial purified protease or cell-free supernatants from cultures of wild-type W81, the chitinase-negative mutant W81M2 or the complemented derivative W81A1 (pCU800) resulted in hyphal lysis and loss of subsequent fungal growth ability once re-inoculated onto fresh plates. In contrast, commercial purified chitinase or cell-free supernatants from cultures of the protease-negative mutant WS1M1 or the chitinase- and protease-negative mutant W81A1 had no effect on integrity of the essentially chitin-free Pythium mycelium, and did not prevent subsequent growth of the fungus. In soil microcosms containing soil naturally infested by Pythium spp., strains W81, W81M2 and W81A1(pCU800) reduced the ability of Pythium spp. to colonize the seeds of sugar beet and improved plant emergence compared with the untreated control, whereas W81A1 and W21M1 failed to protect sugar beet from damping-off. Wild-type W81 and its mutant derivatives colonized the rhizosphere of sugar beet to similar extents, it was concluded that the ability of S. maltophilia W81 to protect sugar beet from Pythium -mediated damping-off was due to the production of an extracellular protease.
Effects of growth-inhibitory concentrations of copper on alginate biosynthesis in highly mucoid Pseudomonas aeruginosa
Authors: Leito Jorge H. , S-Correia Isabel
Published: June 2009

Alginate production and degree of polymerization were affected when the highly mucoid Pseudomonas aeruginosa 8821M was grown with growth-inhibitory concentrations of Cu2+ (supplied as CuCl2; 1-5 mM). The inhibition of alginate biosynthesis was consistent with the decreased activity in Cu2+-stressed cells of phosphomannose isomerase/GDP-mannose pyrophosphorylase (encoded by algA), phosphomannomutase (encoded by algC) and GDP-mannose dehydrogenase (encoded by algD). However, in cells grown with concentrations of CuCl2 below 2 mM, the steady-state mRNA levels from algA, algC, algD and from the regulatory gene algR1 increased moderately. This observation is consistent with the suggested linkage between the control of alginate gene expression and the global regulation involved in the oxidative stress response. At highly inhibitory concentrations the levels of the four alginate gene transcripts decreased from maximal values. The bell-shaped curves, representing the effect of Cu2+ concentration on mRNA levels from the four alginate genes, exhibited similar patterns but did not concur. The decrease of the specific activity of enzymes necessary for GDP-mannuronic acid synthesis in Cu2+-grown cells was correlated with changes in gene expression, with the inhibitory effect of Cu2+ on enzyme activities and with Cu2+-induced oxidative inactivation of enzymes, especially the particularly sensitive phosphomannose isomerase activity.
Properties of NAD+- and NADP+-dependent malic enzymes of Rhizobium (Sinorhizobium) meliloti and differential expression of their genes in nitrogen-fixing bacteroids
Authors: Driscoll Brian T. , Finan Turlough M.
Published: June 2009

The wild-type NAD+-dependent malic enzyme (dme) gene of Rhizobium (now Sinorhizobium) meliloti was cloned and localized to a 3.1 kb region isolated on the cosmid pTH69. This cosmid complemented the symbiotic nitrogen fixation (Fix-) phenotype of R. meliloti dme mutants. The dme gene was mapped by conjugation to between the cys-11 and leu-53 markers on the R. meliloti chromosome. β-Galactosidase activities measured in bacterial strains carrying either dme-lacZ or tme-lacZ gene fusions (the tme gene encodes NADP+-dependent malic enzyme) indicated that the dme gene was expressed constitutively in free-living cells and in N2-fixing bacteroids whereas expression of the tme gene was repressed in bacteroids. The R. meliloti dme gene product (DME) was overexpressed in and partially purified from Escherichia coli. The properties of this enzyme, together with those of the NADP+-dependent malic enzyme (TME) partially purified from R. meliloti dme mutants, were determined. Acetyl-CoA inhibited DME but not TME activity. This result supports the hypothesis that DME, together with pyruvate dehydrogenase, forms a pathway in which malate is converted to acetyl-CoA.
Arginine-, hypoxanthine-, uracil-requiring isolates of Neisseria gonorrhoeae are a clonal lineage within a non-clonal population
Authors: Gutjahr Thorsten S. , O'Rourke Maria , Ison Catherine A. , Spratt Brian G.
Published: June 2009

Multilocus enzyme electrophoresis has shown that a collection of 101 arginine-, hypoxanthine-, uracil-requiring (AHU-) isolates ofNeisseria gonorrhoeae,recovered over a 39 year period from the UK and Denmark, were of a single electrophoretic type (91% of strains), or differed from the predominant electrophoretic type at only a single locus. The striking uniformity of the AHU-isolates, and the correlation between auxotype, serovar and overall genetic background, contrasts with previous studies of gonococcal populations (that included very few AHU-strains), and a small sample of non-AHU-isolates studied here, which demonstrated a non-clonal population structure and a lack of association between auxotype, serovar and genetic background. There was no marked difference in the ability of AHIT isolates to be transformed with their own DNA, or with DNA from gonococci of other auxotypes, and the relative genetic stability of AHU-isolates does not appear to be due to a defect in their ability to be transformed. An alternative possibility is that AHU-gonococci recombine with other lineages, but that the resulting recombinants are not maintained in the population. This would occur, for example, if AHU-gonococci competed poorly in mixed infections, within which effective recombination between lineages occurs, and are usually only transmitted from individuals who are singly infected with an AHU-strain. The association between AHU-gonococci and asymptomatic infections may lead to an increased rate of transmission of these strains which under this scenario would be needed to prevent them from being lost from the population.
O-antigenic determinants in Salmonella species of serogroup C1 are expressed in distinct immunochemical populations of chains
Authors: Nnalue Ndubisi A. , Lindberg Alf A.
Published: June 2009

The O-antigenic specificities found among salmonellae of serogroup C1are O:61,7, O:62,7, O:61,62,7 and O:6,7,14, as defined by classical serology. Factor O:7 is the group-wide determinant while factors O:61,O:62and O:14 are found in some strains but not others. Strains of the O:62,7 specificity are subject to lysogenic conversion by phages 61and 14 to the O:61,7 and O:6,7,14 specificities, respectively. To further delineate antigenic complexity and serological relationships among strains of this serogroup monoclonal antibodies (mAbs) were generated against the O:61,62,7 polysaccharide ofSalmonella thompson.Five mAbs of either the O:61,or the O:62specificities did not bind O:6,7,14 strains or LPS, showing that the O:6 determinant in these strains is neither O:61,nor O:62. Thus antigenic conversion of O:62,7 strains by phage 14 is accompanied by addition of O:14 as well as loss of O:62Three mAbs which demonstrated group-wide reactivity, and were thus specific for O:7, recognized clearly separable epitopes hereby defined as sub-specificities, O:71O:72and O:73Immunoblotting of mAbs against electrophoretically resolved LPS showed that factors O:61and O:62are expressed only in LPS molecules of high molecular mass whereas O:72and O:73are expressed only in relatively low-molecular-mass chains. These results are consistent with the expression of different antigenic determinants in structurally distinct subpopulations of O chains. The implication of the existence of distinct subpopulation of chains is that the published structure of the O:6,7 repeat unit is not fully representative of the O-antigenic structure of this group.
Dimethyl sulphoxide reduction with reduced sulphur compounds as electron donors by anoxygenic phototrophic bacteria
Authors: Vogt Carsten , Rabenstein Andreas , Rethmeier Jrg , Fischer Ulrich
Published: June 2009

The marine purple non-sulphur bacterium Rhodovulum euryhalinum strain DSM 4868 reduced dimethyl sulphoxide (DMSO) to dimethyl sulphide (DMS) chemotrophically with sulphide as electron donor. The oxidation of sulphide was correlated with the formation of polysulphides. R. euryhalinum reduced DMSO phototrophically with sulphide as well, but the amount of DMSO reduced in relation to sulphide oxidized was lower. The marine green sulphur bacterium Chlorobium vibrioforme strain DSM 8327 reduced DMSO to DMS phototrophically with sulphide and thiosulphate as electron donors. The extent of DMSO reduction was much less in the dark. Eight strains of purple sulphur bacteria - marine, brackish water and freshwater isolates - and another marine green sulphur bacterium showed a very weak capacity for DMSO reduction with sulphide or thiosulphate as electron donors in the light and dark, respectively.
Overlapping promoters modulate Fnr- and ArcA-dependent anaerobic transcriptional activation of the focApfl operon in Escherichia coli
Authors: Kaiser Manuela , Sawers Gary
Published: June 2009

The recently identified P6A promoter of the anaerobically inducible focApfl operon of Escherichia coli overlaps the Fnr (fumarate-nitrate reduction regulator)-dependent P6 promoter. The Fnr-binding site of P6 and the -35 hexamer sequence of P6A are shared between the promoters. Inactivation of P6A, through introduction of a -10 hexamer mutation, resulted in enhanced anaerobic induction of operon expression. The dependence on the ArcA (aerobic respiration control regulator) and Fnr transcription factors for anaerobic induction was tested for several focA-lacZ and pfl-lacZ gene fusions. Anaerobic induction became more dependent on Fnr in derivatives lacking a functional P6A promoter compared with wild-type constructs. Moreover, aerobic expression of the focA gene was reduced by the p6A mutation, as was the dependence on ArcA for anaerobic induction. Inactivation of P6 severely reduced Fnr-dependent anaerobic induction, in accord with previous findings. Transcription analyses demonstrated that a mutation in the -10 hexamer sequence of either P6A or P6 did not adversely affect transcription from the remaining promoter. Taken together, these results indicate that the P6A promoter moderates the Fnr-dependent activation of P6 through competition for RNA polymerase binding.
Expression of the mau gene cluster of Paracoccus denitrificans is controlled by MauR and a second transcription regulator
Authors: Delorme CéAcile , Huisman Tako T. , Reijnders Willem N. M. , Chan Yin-Lay , Harms Nellie , Stouthamer Adriaan H. , van Spanning Rob J. M.
Published: June 2009

The mau gene cluster of Paracoccus denitrificans constitutes 11 genes (10 are located in the transcriptional order mauFBEDACJGMN; the 11th, mauR, is located upstream and divergently transcribed from these genes) that encode a functional methylamine-oxidizing electron transport branch. The mauR gene encodes a LysR-type transcriptional activator essential for induction of the mau operon. In this study, the characteristics of that process were established. By using lacZ transcriptional fusions integrated into the genome of P. denitrificans, it was found that the expression of the mauR gene during growth on methylamine and/or succinate was not autoregulated, but proceeded at a low and constant level. The mauF promoter activity was shown to be controlled by MauR and a second transcriptional regulator. This activity was very high during growth on methylamine, low on succinate plus methylamine, and absent on succinate alone. MauR was overexpressed in Escherichia coli by using a T7 RNA polymerase expression system. Gel shift assays indicated that MauR binds to a 403 bp DNA fragment spanning the mauR-mauF promoter region. It is concluded from these results that the expression of the structural mau genes is dependent on MauR and its inducer, methylamine, as well as on another transcription factor. Both activators are required for high-level transcription from the mauF promoter. It is hypothesized that the two activators act synergistically to activate transcription: the effects of the two activators are not additive and either one alone activates the mauF promoter rather weakly.
Reversion rates in a leuB auxotroph of Escherichia coli K-12 correlate with ppGpp levels during exponential growth
Authors: Wright Barbara E. , Minnick Michael F.
Published: June 2009

Two isogenic strains of Escherichia coli K-12 differing only in relA, as well as two spoT transductants of the relA
-strain, were examined with respect to ppGpp levels and reversion rates of a leuB
-allele under nine different conditions. A positive correlation was established between reversion rates and the steady-state concentration of ppGpp during exponential growth. The leuB genes from two leuB
-strains (isogenic except for relA) were cloned and sequenced and found to contain a single mutation, namely, a C-to-T transition at nucleotide 857. This mutation resulted in a serine-to-leucine substitution at amino acid residue 286 of the LeuB protein. PCR products that encompassed the leuB lesion were generated from 53 revertants and then sequenced. Of these revertants, 36 were found to contain nucleotide substitutions that would result in a serine (wild type), valine or methionine at amino acid residue 286 of LeuB, and nearly all of them exhibited generation times similar to wild type. Seventeen of the analysed revertants were found to be suppressors that retained the encoded leucine at residue 286. The majority of the suppressor mutants exhibited generation times that were significantly longer than wild type.
Squalene-hopene cyclase from Bradyrhizobium japonicum: cloning, expression, sequence analysis and comparison to other triterpenoid cyclases
Authors: Perzl Michael , Müller Peter , Poralla Karl , Kannenberg Elmar L.
Published: June 2009

With the help of a PCR-based screening method, the gene encoding squalene-hopene cyclase (SHC) ofBradyrhizobium japonicumUSDA 110 was isolated from a cosmid library. The SHC catalyses the cyclization of squalene to hopanoids, a class of triterpenoid lipids recently discovered in nitrogen-fixing, root-nodule-formingBradyrhizobiumbacteria. Hybridization experiments showed that the gene is present in bacteria of allBradyrhizobiumstrains tested and in photosynthetic bacteria forming stem nodules on tropical legumes of the genusAeschynomene.TheBradyrhizobium shcgene is 1983 bp in length and encodes a protein of 660 amino acid residues with a calculated molecular mass of 73671 Da. Comparison of the deduced amino acid sequence with the sequences of other SHCs revealed highest similarity (70%) to the SHC from the Gram-negativeZymomonas mobilisand lower similarity (48%) to the SHCs from the Gram-positiveAlicyclobacillus acidocaldariusandAlicyclobacillus acidoterrestris. BradyrhizobiumSHC also showed similarity (38-43%) to eukaryotic oxidosqualene cyclases. TheB. japonicum shcgene was expressed inEscherichia coli.The recombinant SHC catalysed the cyclization of squalene to the hopanoids hopene and diplopterolin vitro.However, the formation of the gammacerane derivative tetrahymanol, which is produced in addition to hopanoids inB. japonicumstrainsin vivo,could not be detectedin vitro.Therefore, the presence of a second squalene cyclase inB. japonicumcan be assumed. Sequence analysis of 0.5 kb upstream from theshcgene identified a partial ORF with significant similarity to the C-terminus of an ORF located immediately upstream from theshcgene inZ. mobilis.Both ORFs also showed similarity to phytoene desaturases from cyanobacteria and plants. The 3'-end of this ORF fromB. japonicumoverlaps with 13 bp at the 5'-end ofshc.The close proximity of this ORF toshcsuggests thatshcand this ORF may be part of an operon.
The temperature sensitivity of Bacillus subtilis DB1005 is due to insufficient activity, rather than insufficient concentration, of the mutant σA factor
Authors: Chang Ban-Yang , Liao Chao-Tsai , Wen Yu-Der , Wang Wen-Horng
Published: June 2009

The σA factor of Bacillus subtilis DB1005 contains two amino acid substitutions (1198A and 1202A) in the promoter –10 binding region. It has been confirmed that this σ factor is responsible for the temperature sensitivity of B. subtilis DB1005. An investigation was conducted into how the mutantσA could cause temperature-sensitive (Ts) cell growth by analysing its structural stability, cellular concentration and transcriptional activity. The mutant σA was unstable even at the permissive temperature of 37°C (t1/2 59 min), whereas the wild-type counterpart was fairly stable under the same conditions (t
1/2 600 min). However, neither wild-type σA nor mutant σA was stable at 49°C (t
1/2 34 min and 23 min, respectively). Analyses of the rates of σA synthesis revealed that B. subtilis DB1005 was able to compensate for unstable σ by elevating the level of σA at 37°C but not at 49°C. Moreover, overexpression of the mutant σA at 49°C could not suppress the Ts phenotype of B. subtilis DB1005. This indicates that the temperature sensitivity of B. subtilis DB1005 is not due to insufficient σA concentration in the cell. The greater decline of an already reduced activity of the mutant σA at 49°C suggests that the temperature sensitivity of B. subtilis DB1005 is instead the result of a very low activity of σ A probably below a critical level necessary for cell growth.
Characterization of polar-flagellar-length mutants in Vibrio alginolyticus
Authors: Furuno Masaaki , Atsumi Tatsuo , Yamada Taku , Kojima Seiji , Nishioka Noriko , Kawagishi Ikuro , Homma Michio
Published: June 2009

Vibrio alginolyticus has two types of flagella, polar (Pof) and lateral (Laf). From a Laf-defective mutant (Pof+Laf-), polar-flagellar-length mutants which have short Pof and long Pof were isolated. The mean lengths of the helical axis in wild-type, short and long Pof were 5.5.0.9 μm, 2.5.0.6 μm and 11.2.3.6 μm, respectively. The swimming speeds of the short- and long-Pof mutants were slower than that of the wild-type strain. The relationship between swimming speed and flagellar length in a population of mutant cells was examined. In the short-Pof mutant, the decrease of swimming speed seemed to be derived from the decrease in flagellar length. In the long-Pof mutant, there was almost no correlation between swimming speed and flagellar length, and the slow swimming was explained by the helical shape of the flagella, whose pitch and radius were 1.4 μm and 0.062 μm, respectively, whereas those of the wild-type flagella were 1.5 μm and 0.16 μm. The relative amounts of the various molecular components of the long Pof were different from those of the wild-type or the short Pof. This seems to be the reason for the difference in flagellar shape and length, though the mutation may be pleiotropic and affect flagellar function or regulation.
Increased pyruvate orthophosphate dikinase activity results in an alternative gluconeogenic pathway in Rhizobium (Sinorhizobium) meliloti
Authors: Magne østerås , Driscoll Brian T. , Finan Turlough M.
Published: June 2009

The formation of phosphoenolpyruvate (PEP) is a major step in the gluconeogenic pathway in which tricarboxylic acid (TCA) cycle intermediates are converted to hexose sugars. In Rhizobium (now Sinorhizobium) meliloti this step is catalysed by the enzyme PEP carboxykinase (PCK) which converts oxaloacetate to PEP. R. meliloti Pck-mutants grow very poorly with TCA cycle intermediates as the sole source of carbon. Here, the isolation and mapping of suppressor mutations which allow Pck-mutants to grow on succinate and other TCA cycle intermediates is reported. Tn5 insertions which abolished the suppressor phenotype and mapped to the suppressor locus were located within the pod gene encoding pyruvate orthophosphate dikinase (PPDK). Strains carrying suppressor mutations had increased PPDK activity compared to the wild-type. The suppressor phenotype was dependent on the combined activities of malic enzyme and PPDK, which thus represent an alternative route for the formation of PEP in R. meliloti. PPDK activity was not required for symbiotic N2 fixation.
Control of Neisseria gonorrhoeae pilin gene expression by environmental factors: involvement of the pilA/pilB regulatory genes
Authors: Larribe Mireille , Taha Muhamed-Kheir , Topilko Andrzej , Marchal Christian
Published: June 2009

The control of the expression of the pilin gene (pilE) in Neisseria gonorrhoeae under a wide variety of growth conditions has been studied. The expression of pilE was measured using transcriptional fusions between pilE and the gene encoding chloramphenicol acetyltransferase (CAT), and the level of pilin production was measured by Western blot analysis. Many of the conditions tested affected both growth rate and pilin gene expression (e.g. isoleucine, high osmolarity, high temperature, anaerobic growth, pH 6, urea and iron depletion). Changes in the level of many other proteins were also observed, depending on the conditions, indicating that gonococci undergo an adaptive response to environmental variations. Moreover, environment-induced changes in the level of many proteins, including pilin, seem to involve the pilA/pilB regulatory system, which has been previously proposed to modulate the expression of the gonococcal pilin gene.
Adaptation of proteases and carbohydrases of saprophytic, phytopathogenic and entomopathogenic fungi to the requirements of their ecological niches
Authors: Leger Raymond J. St , Joshi Lokesh , Roberts Donald W.
Published: June 2009

The abilities of isolates of saprophytes (Neurospora crassa, Aspergillus nidulans), an opportunistic human pathogen (Aspergillus fumigatus), an opportunistic insect pathogen (Aspergillus flavus), plant pathogens (Verticillium albo-atrum, Verticillium dahliae, Nectria haematococca), a mushroom pathogen (Verticillium fungicola) and entomopathogens (Verticillium lecanii, Beauveria bassiana, Metarhizium anisopliae) to utilize plant cell walls and insect cuticle components in different nutrient media were compared. The pathogens showed enzymic adaptation to the polymers present in the integuments of their particular hosts. Thus, the plant pathogens produced high levels of enzymes capable of degrading pectic polysaccharides, cellulose and xylan, as well as a cutinase substrate, but secreted little or no chitinase and showed no proteolytic activity against elastin and mucin. The entomopathogens and V. fungicola degraded a broad spectrum of proteins (including elastin and mucin) but, except for chitinase, cellulase (V. lecanii and V. fungicola only) and cutinase (B. bassiana only), produced very low levels of polysaccharidases. The saprophytes (Neu. crassa and A. nidulans) and the opportunistic pathogens (A. fumigatus and A. flavus) produced the broadest spectrum of protein and polysaccharide degrading enzymes, indicative of their less specialized nutritional status. V. lecanii and V. albo-atrum were compared in more detail to identify factors that distinguish plant and insect pathogens. V. albo-atrum, but not V. lecanii, grew well on different plant cell wall components. The major class of proteases produced in different media by isolates of V. albo-atrum and V. dahliae were broad spectrum basic (pl > 10) trypsins which degrade Z-AA-AA-Arg-NA substrates (Z, benzoyl; AA, various amino acids; NA, nitroanilide), hide protein azure and insect (Manduca sexta) cuticles. Analogous peptidases were produced by isolates of V. lecanii and V. fungicola but they were specific for Z-Phe-Val-Arg-NA. V. albo-atrum and V. dahliae also produced low levels of neutral (pl ca 7) and basic (pl ca 9.5) subtilisin-like proteases active against a chymotrypsin substrate (Succinyl-Ala2-Pro-Phe-NA) and insect cuticle. In contrast, subtilisins comprised the major protease component secreted by V. lecanii and V. fungicola. Both V. lecanii and V. albo-atrum produced the highest levels of subtilisin and trypsin-like activities during growth on collagen or insect cuticle. Results are discussed in terms of the adaptation of fungi to the requirements of their ecological niches
Repression and inactivation of α-amylase in Thermomonospora species during growth on cellobiose
Authors: Busch J. E. , Stutzenberger F. J.
Published: June 2009

Thermophilic actinomycetes establish themselves as numerically dominant bacterial populations in selected high temperature environments by virtue of their exoenzymic ability to degrade the complex polysaccharides in thermogenic plant biomass. When Thermomonospora curvata and Thermomonospora fusca were grown on a mixture of cellulose and starch in mineral salts minimal medium, α-amylase was repressed via inhibition of maltose uptake by cellobiose. Addition of cellobiose to exponential phase cells growing on maltose or maltotriose triggered rapid degradation of extant amylase in the culture fluid of wild-type cells, but not in a protease-deficient mutant of T. fusca. A serine protease purified from T. fusca caused inactivation of the amylase in culture fluid of the mutant when added at a concentration approximating to that of the wild-type strain. The chelating agent, EDTA, accelerated inactivation by the protease, while the presence of calcium or amylase reaction products protected the amylase. Therefore, during growth in an environment containing multiple polysaccharides, these thermophiles control the levels of their extracellular depolymerizing enzymes via both inducer exclusion and proteolytic inactivation.
Metabolic regulation of Irp gene expression in Escherichia coli K-12
Authors: Chen C. F. , Lan J. , Korovine M. , Shao Z. Q. , Tao L. , Zhang J. , Newman E. B.
Published: June 2009

Expression of the Irp gene is regulated in part by the nutrients available to the cell, and is decreased in rich medium, in glucose minimal media enriched with amino acids, and in minimal medium with alternative carbon sources, such as acetate and succinate. When Lrp production is increased in a given medium, expression of its target genes is also increased. However, when the medium is changed from glucose to acetate, the response of the target genes is governed by many factors.
Involvement of the gonococcal MtrE protein in the resistance of Neisseria gonorrhoeae to toxic hydrophobic agents
Authors: Delahay R. M. , Robertson B. D. , Balthazar J. T. , Shafer W. M. , Ison C. A.
Published: June 2009

Summary: Low-level resistance of Neisseria gonorrhoeae to toxic hydrophobic agents (HAs), including some antibiotics, is chromosomally mediated via the multiple transferable resistance (mtr) efflux system. The gene encoding the 48.3 kDa outer-membrane protein MtrE, which is associated with the mtr phenotype, was identified and is homologous to export-associated outer-membrane proteins, including the OprM (formerly OprK) lipoprotein of Pseudomonas aeruginosa. Insertional inactivation of the mtrE gene in N. gonorrhoeae strain FA19 resulted in the loss of the outer-membrane protein, with concomitant hypersusceptibility of the mutant strain to a range of HAs. The properties of this mutant confirmed the role of MtrE in multidrug resistance mediated by an active efflux mechanism. Secondary structure predictions for MtrE indicated a largely hydrophilic protein with a single α-helical transmembrane region. A transposon-like element, similar to that found downstream of the region containing the promoters for mtrR and mtrC in Neisseria meningitidis, was identified 63 bp downstream of the mtrE gene.
Divergence and conservation of the partitioning and global regulation functions in the central control region of the IncP plasmids RK2 and R751
Authors: Macartney Donia P. , Williams D. Ross , Stafford Theresa , Thomas Christopher M.
Published: June 2009

Summary: The central control region (Ctl) of IncP plasmids is associated with two phenotypes: the coordinate expression of replication and transfer genes; and the ability to increase the segregational stability of a low-copy-number test plasmid. This region of the IncP plasmid R751 shows significant sequence divergence from the IncPα plasmid RK2 sequence, and two genes, korF and korG, present in the IncPα region are missing in the IncP Ctl. In other respects the organization of the Ctl is basically the same. Although the two key global regulatory genes korA and korB are highly conserved, studies on their ability to repress transcription from a variety of IncPα and IncP plasmid promoters suggest differences in operator recognition by KorA and synergy with other repressors. The products of kfrA, upf54.8 and upf54.4 genes are conserved; KfrA shows least conservation and, while retaining the ability to act as a transcriptional repressor, appears to have completely different DNA-binding specificity. The genes required for the plasmid segregational stabilization (partitioning) phenotype - incC, korB and the KorB operator OB3 - are conserved and contribute to a more efficient plasmid stabilization than the IncPα equivalents. This may indicate that the Ctl plays an especially important role in partitioning of IncP plasmids, since they lack the second stability region (parlmrs) found in IncP plasmids.
A Pneumocystis carinii multi-gene family with homology to subtilisin-like serine proteases
Authors: Lugli Elena B. , Allen Andrew G. , Wakefield Ann E.
Published: June 2009

Summary: Copies of a multi-gene family, named PRT1 (protease 1, encoding a subtilisin-like serine protease were cloned from the opportunistic fungal pathogen Pneumocystis carinii. Comparison of the nucleotide sequence of a genomic clone and a cDNA clone of PRT1 from P. carinii f. sp. carinii revealed the presence of seven short introns. Several different domains were predicted from the deduced amino acid sequence: an N-terminal hydrophobic signal sequence, a pro-domain, a subtilisin-like catalytic domain, a P-domain (essential for proteolytic activity), a proline-rich domain, a serine/threonine-rich domain and a C-terminal hydrophobic domain. The catalytic domain showed high homology to other eukaryotic subtilisin-like serine proteases and possessed the three essential residues of the catalytic active site. Karyotypic analysis showed that PRT1 was a multi-gene family, copies of which were present on all but one of the P. carinii f. sp. carinii chromosomes. The different copies of the PRT1 genes showed nucleotide sequence heterogeneity, the highest level of divergence being in the proline-rich domain, which varied in both length and composition. Some copies of PRT1 were contiguous with genes encoding the P. carinii major surface glycoprotein.
An outer-membrane porin inducible by short-chain amides and urea in the methylotrophic bacterium Methylophilus methylotrophus
Authors: Mills James , Greenwood Jacqueline A. , Wyborn Neil R. , Williams Steven G. , Jones Colin W.
Published: June 2009

Summary: ThefmdAandfmdBgenes encoding formamidase and a putative regulatory protein, respectively, from the methylotrophic bacteriumMethylophilus methylotrophuswere recloned with additional flanking DNA (pSW1).fmdC,encoding a weakly hydrophilic protein containing an N-terminal signal sequence, was identified upstream offmdAB.The derived amino acid sequence of mature FmdC (Mr39204) showed that it was rich in -sheet and aromatic amino acids, and exhibited significant similarities to several outer-membrane porins from other bacteria. Cell fractionation studies showed that the protein was located in the outer membrane. Mature FmdC was purified and shown to consist of a single type of subunit (Mr40000) with the predicted N-terminal amino acid sequence (GATISF-). SDS-PAGE and Western blotting of cells grown in continuous culture under various conditions showed that mature FmdC was induced by formamide, acetamide and urea, repressed by excess ammonia, and over-expressed during prolonged growth under formamide limitation. It is concluded that mature FmdC is a porin involved in the transport of short-chain amides and urea through the outer membrane ofM. methylotrophusunder conditions where these nitrogen sources are present at very low concentration.
Protein kinase Sck1 is involved in trehalase activation by glucose and nitrogen source in the fission yeast Schizosaccharomyces pombe
Authors: Soto T. , Fernandez J. , Cansado J. , Vicente-Soler J. , Gacto M.
Published: June 2009

Summary: Trehalase activity is markedly enhanced upon addition of glucose and a nitrogen source to cells of the fission yeastSchizosaccharomyces pombe.This increase corresponds to a post-translational activation of the enzyme, which is controlled by cAMP-dependent and cAMP-independent pathways. Recent work has shown that overexpression of SCK1 in Schiz. pombe is able to suppress mutations that result in reduced Pka1 (cAMP-dependent protein kinase A activity, suggesting that Sck1 (suppressor of loss of cAMP-dependent protein kinase) might be a functional analogue of Pka1 in the fission yeast. Here, an analysis of the possible role of Sck1 in the activation of trehalase triggered by glucose and a nitrogen source is reported in cells that were deficient in either Pka1, Sck1 or both protein kinases. The results showed that, except in repressed cells, Sck1 probably mediates a cAMP-independent activation of trehalase following the signal(s) triggered by glucose and the nitrogen source. The absence of functional Sck1 in derepressed cells renders trehalase insensitive to activation by glucose and the nitrogen source even in the presence of Pka1, indicating that the Sck1-dependent, cAMP-independent pathway is the main signalling pathway controlling trehalase activation under derepression conditions. It is proposed that, during the activation of trehalase induced by glucose or a nitrogen source, the cAMP-Pka1 activation pathway previously characterized is to some extent parallel to this newly described one which includes Sck1 as ohosphorylating enzyme. Neither of these two pathways, however, plays a key role in the heat-induced increase in trehalase activity.
Tn5041: a chimeric mercury resistance transposon closely related to the toluene degradative transposon Tn4651
Authors: Kholodii G. Ya. , Yurieva O. V. , Gorlenko Zh. M. , Mindlin S. Z. , Bass I. A. , Lomovskay O. L. , Kopteva A. V. , Nikiforov V. G.
Published: June 2009

Summary: This paper reports the discovery and characterization of Tn5041, a novel-type transposon vehicle for dissemination of mercury resistance in natural bacterial populations. Tn5041 (14876 bp), identified in a Pseudomonas strain from a mercury mine, is a Tn3 family mercury resistance transposon far outside the Tn21 subgroup. As in other Tn3 family transposons, Tn5041 duplicates 5 bp of the target sequence following insertion. Tn5041 apparently acquired its mer operon as a single-ended relic of a transposon belonging to the classical mercury resistance transposons of the Tn21 subgroup. The putative transposase and the 47 bp terminal inverted repeats of Tn5041 are closely related to those of the toluene degradative transposon Tn4651 and fall into a distinct subgroup on the fringe of the Tn3 family. The amino acid sequence of the putative resolvase of Tn5041 resembles site-specific recombinases of the integrase family. Besides the mer operon and putative transposition genes, Tn5041 contains a 4 kb region that accommodates a number of apparently defective genes and mobile elements.
DNA polymorphism at the omp-31 locus of Brucella spp.: evidence for a large deletion in Brucella abortus, and other species-specific markers
Authors: Vizcaíno Nieves , Verger Jean-Michel , Grayon Maggy , Zygmunt Michel S. , Cloeckaert Axel
Published: June 2009

Summary: The omp-31 gene, encoding a major outer-membrane protein in Brucella melitensis, was PCR-amplified from Brucella strains representing all species and known biovars by using primers selected according to the B. melitensis 16M omp-31 published sequence. Amplification of omp-31 was achieved from DNA of all Brucella species with the exception of Brucella abortus, the only Brucella species where expression of omp-31 was not detected by reactivity with an mAb specific for an epitope located in Omp-31. Southern blot hybridization of plasmid probes, bearing inserts (4.4-17 kb) containing B. melitensis 16M omp-31 and adjacent DNA of different sizes, with HindIII-digested total DNA showed that a large fragment, comprising the entire omp-31 gene and flanking DNA, was actually absent in B. abortus strains. The size of this DNA fragment has been determined to be about 10 kb. Southern blot hybridization with the different plasmid probes identified species-specific markers for B. abortus and B. melitensis. At the biovar level, a specific marker for B. melitensis bv. 1 was also identified. Additionally, PCR-RFLP studies of omp-31 revealed specific markers for Brucella ovis, Brucella canis and Brucella suis bv. 2. Using a combination of omp-31 PCR-RFLP patterns and Southern blot hybridization profiles Brucella species were differentiated with the sole exception of Brucella neotomae which was not differentiated from B. suis bv. 1, 3, 4 and 5. Results presented in this paper demonstrate the potential of omp-31 for differentiating the brucellae and show that B. abortus lacks a large DNA fragment of about 10 kb containing omp-31 and flanking DNA. In such a large deletion, other genes in addition to omp-31 are probably involved. Sequencing of this DNA fragment will help to identify the missing genes in B. abortus which could possibly be involved in the differences of pathogenicity and host preference seen in Brucella species.
Elucidation of the metabolic pathway for dibenzothiophene desulphurization by Rhodococcus sp. strain IGTS8 (ATCC 53968)
Authors: Oldfield Christopher , Pogrebinsky Olga , Simmonds Julie , Olson Edwin S. , Kulpa Charles F.
Published: June 2009

Summary: Rhodococcus sp. strain IGTS8 (ATCC 53968) is able to utilize dibenzothiophene (DBT) as a sole source of sulphur. The carbon skeleton of DBT is not metabolized and is conserved as 2-hydroxybiphenyl (HBP), which accumulates in the medium. This phenotype is due to the expression of the plasmid-encoded DBT-desulphurization (dsz) operon, which encodes three proteins, DszA, B and C. In this paper it is shown, using [35S]DBT radiolabelling studies, that sulphur is released in the form of inorganic sulphite. The pathway of DBT desulphurization is described in detail. In summary, DszC catalyses the stepwise S-oxidation of DBT, first to dibenzothiophene 5-oxide (DBTO) and then to dibenzothiophene 5,5-dioxide (DBTO2); DszA catalyses the conversion of DBTO2 to 2-(2′-hydroxyphenyl)benzene sulphinate (HBPSi−) and DszB catalyses the desulphination of HBPSi− to give HBP and sulphite. Studies with cell-free extracts show that DszA and DszC, but not DszB, require NADH for activity. 18O2-labelling studies show that each incorporated oxygen atom is derived directly from molecular oxygen. These results are consistent with the role of DszC as a mono-oxygenase, of DszA as an apparently unique enzyme which catalyses the reductive hydroxylation of DBTO2 leading to cleavage of the thiophene ring, and of DszB as an aromatic sulphinic acid hydrolase.
Extracellular proteases produced by the Quorn® myco-protein fungus Fusarium graminearum in batch and chemostat culture
Authors: Griffen Alison M. , Wiebe Marilyn G. , Robson Geoffrey D. , Trinci Anthony P. J.
Published: June 2009

Summary: Fusarium graminearum was grown in batch and continuous (chemostat) culture on a glucose-mineral salts medium in the presence and absence of casein. In the absence of casein no protease activity was detected in the culture filtrate from either batch or chemostat culture. For batch cultures grown on medium containing casein, most of the proteolytic activity detected in the supernatant during exponential growth had an optimum at ca pH 5.0. However, as the cultures passed from late exponential into stationary phase, the pH profile of the protease activity broadened until most of it was in the alkaline pH region. For glucose-limited chemostat cultures grown on media containing casein, protease activity had a narrow pH optimum with maximum activity at pH 5.0. For all concentrations of casein examined, protease activity was greater in chemostat culture than in batch culture. Extracellular proteases from batch and chemostat cultures were purified by bacitracin-Sepharose affinity chromatography. At least seven proteins were purified from batch cultures but chemostat cultures contained only a single aspartic protease with a molecular mass of 40 kDa.
Cell wall protein mannosylation determines Candida albicans cell surface hydrophobicity
Authors: Masuoka James , Hazen Kevin C.
Published: June 2009

Summary: Cell surface hydrophobicity (CSH) has been shown to be an important factor in the ability of the opportunistic pathogenic yeast Candida albicans to adhere to surfaces. Hydrophobic cells adhere more readily to host tissue, and are more resistant to phagocytic killing, than hydrophilic cells. Consequently, CSH plays an important role in the pathogenicity of C. albicans. Previous work suggested a relationship between CSH and cell wall protein glycosylation. The present work tests the hypothesis that changes in outer chain mannosylation, rather than complete loss of oligosaccharide groups, are sufficient to modulate CSH. These studies compared wild-type cells to a variant that has altered mannosylation and is hydrophobic under conditions in which wild-type cells are hydrophilic. Composition analysis of cell surface digests showed that the glycosylation of wild-type cell surface proteins was much more extensive than that seen in the variant. Antibodies which recognize the acid-labile and acid-stable portions of C. albicans mannan showed not only differences between wild-type and variant cells but also differences between wild-type hydrophilic and wild-type hydrophobic cells. The results suggest that exposure of surface hydrophobic regions on C. albicans may be related to the abundance of phosphodiester-linked, acid-labile mannosyl groups rather than the complete loss of outer chain mannosylation on cell wall proteins.
A Candida albicans RAS-related gene (CaRSRl) is involved in budding, cell morphogenesis and hypha development
Authors: Yaar Liora , Mevarech Moshe , Koltint Yigal
Published: June 2009

Summary: Candida albicans, the most important human fungal pathogen, is a dimorphic fungus that can grow either as a yeast or as a hyphal form in response to medium conditions. A RAS-related C. albicans gene (CaRSRl) was isolated as a suppressor of a cdc24b bud-emergence mutation of the baker's yeast, Saccharomyces cerevisiae. The deduced protein encoded by CaRSRl is 248 amino acids long and 56% identical to that encoded by the 5. cerevisiae RSRl (BUDI) gene. Disruption of CaRSRl in C. albicans indicated that CaRSRl is involved in both yeast and hypha development. In the yeast phase, CaRSRl is required for normal (polar) bud site selection and is involved in cell morphogenesis; in the yeast-mycelial transition it is involved in germ tube emergence; and in the development of the hyphae it is involved in cell elongation. The disruption of CaRSRl leads to reduced virulence in both heterozygote and homozygote disruptants in a dose-dependent manner. The reduced virulence can be attributed to the reduced germination and shorter hyphae resulting from the disruption of CaRSRl.
Mutant studies of phosphofructo-2-kinases do not reveal an essential role of fructose-2, 6-bisphosphate in the regulation of carbon fluxes in yeast cells
Authors: Müller Susanne , Zimmermann Friedrich K. , Boles Eckhard
Published: June 2009

Summary: The effect of the allosteric regulator fructose-2, 6-bisphosphate (F2, 6bP) on the regulation of carbohydrate metabolism was investigatedin vivowithSaccharomyces cerevisiaemutants containing no, very high or unregulated 6-phosphofructo-2-kinase activity. Simultaneous overproduction of F2, 6bP and 6-phosphofructo-1-kinase activity did not increase the glycolytic flux to ethanol. Overexpression of fructose-1, 6-bisphosphatase during growth on glucose in a mutant strain devoid of F2, 6bP did not cause pronounced effects on the cells. Moreover, high levels of F2, 6bP during growth on ethanol in a strain with a highly active 6-phosphofructo-2-kinase enzyme did not affect either carbon flux to glycogen or growth rate. Site-directed mutagenesis of 6-phosphofructo-2-kinase (Pfk26) revealed that serine 644 is involved in the activation of Pfk26 by protein kinase A phosphorylation, but that, additionally, the enzyme can be further activated by phosphorylation of another amino acid residue. The results demonstrate that F2, 6bP is not needed to sustain an adequate glycolytic flux under fermentative conditions, but rather is concerned with the homeostasis of metabolite concentrations. Moreover, they fail to indicate a physiological significance for inhibition of fructose-1,6-bisphosphatase by F2,6bP.
Physical identification of a chromosomal locus encoding biosynthetic genes for the lipopeptide calcium-dependent antibiotic (CDA) of Streptomyces coelicolor A3(2)
Authors: Chong Pei Pei , Podmore Sylvia M. , Kieser Helen M. , Redenbach Matthias , Turgay Kürsad , Marahiel Mohamed , Hopwood David A. , Smith Colin P.
Published: June 2009

Summary: Putative peptide-synthetase-encoding DNA fragments were isolated from theStreptomyces coelicolorA3(2) chromosome using a PCR-based approach and mapped to a single ∼ 35 kb segment. In integrative transformation experiments, DNA fragments from this region disrupted production of the calcium-dependent antibiotic (CDA) and had sequences characteristic of non-ribosomal peptide synthetases, thus proving that thecdalocus had been cloned.