bluuebunny/crossref_metadata_2025 huggingface dataset:
Molecular analysis and expression of the extracellular lipase of Aeromonas hydrophila MCC-2
Authors: Chuang Yin Ching , Chiou Shu Fen , Su Jer Horng , Wu Mei Li , Chang Ming Chung
Published: June 2009

The structural gene encoding the extracellular lipase of Aeromonas hydrophila MCC-2 was cloned and found to be expressed in Escherichia coli using its own promoter. When the cloned gene (lip) was expressed in E. coli minicells, an 80 kDa protein was identified. Subcellular fractionation of E. coli carrying the lip gene indicated that the Lip protein was mainly associated with the membrane fraction. Nucleotide sequence analysis revealed that the gene is 2253 bp long, coding for a 79.9 kDa protein with an estimated pl of 10.36. The deduced protein contains two putative signal peptide cleavage sites; one is a typical signal peptidase cleavage site and the other bears a strong resemblance to known lipoprotein leader sequences. Radioactivity from [3H]palmitate was incorporated into the Lip protein when expressed in E. coli. The deduced protein contains a sequence of VHFLGHSLGA which is very well conserved among lipases. It shows 67% and 65% overall identity to the amino acid sequences of lipase from A. hydrophila strains H3 and JMP636, respectively, but shows little homology to those of other lipases. The Lip protein was purified to homogeneity from both A. hydrophila and recombinant E. coli. In hydrolysis of p-nitrophenyl esters and triacylglycerols, using purified enzyme, the optimum chain lengths for the acyl moiety on the substrate were C10 to C12 for ester hydrolysis and C8 to C10 for triacylglycerol hydrolysis.
Primary and secondary structures of rRNA spacer regions in enterococci
Authors: Naïmi Afaf , Beck Geneviéve , Branlant Christiane
Published: June 2009

The 16S-23S and 23S-5S rRNA spacer DNA regions (spacer regions 1 and 2, respectively) from Enterococcus faecalis, Enterococcus faecium, Enterococcus hirae, Enterococcus durans and Enterococcus mundtii were amplified by PCR. Their nucleotide sequences were established and a secondary structure model showing the interaction between the two spacer regions was built. Whereas lactococci and Streptococcus sensu stricto are characterized by a single type of spacer region 1, the enterococci show a high degree of variability in this region; thus the spacer regions 1 with and without tRNAAlawere characterized. However, as shown for lactococci and Streptococcus sensu stricto, the tRNAAlagene does not encode the 3'-terminal CCA trinucleotide. A putative antitermination signal is found downstream from the tRNAAlagene. Based on comparison with Lactococcus lactis and Streptococcus thermophilus, a double-stranded processing stem is proposed. In E. hirae, one of the three different types of spacer region 1 contains no tRNAAla, but displays a 107 nt insertion that forms a long stem-loop structure. A similar insertion (115 nt in length) was found in E. faecium and base compensatory mutations preserve the ability to form the long stem-loop structure. Such insertions may correspond to mobile intervening sequences, as found in the 23S rRNA coding sequences of some Gram-negative bacteria. The spacer regions 1 and 2 from the three subgroups of streptococci were compared, and except for the tRNAAlagene and the double-stranded processing sites, little similarity was found, which opens large possibilities for future development of DNA-based typing methods.
Characterization of Mycobacterium tuberculosis LexA: recognition of a Cheo (Bacillus-type SOS) box
Authors: Movahedzadeh Farahnaz , Colston M. Joseph , Davis Elaine O.
Published: June 2009

The gene coding for the Mycobacterium tuberculosis homologue of LexA has been cloned and sequenced. Amino acids required for autocatalytic cleavage are conserved, whereas those important for specific DNA binding are not, when compared with Escherichia coli LexA. The transcriptional start site was mapped and a DNA sequence motif was identified which resembled the consensus Cheo box sequence involved in the regulation of DNA-damage-inducible genes in Bacillus subtilis. The M. tuberculosis LexA protein was overexpressed in E. coli and purified by means of a His tag. The purifed LexA was shown to bind to the Cheo box sequence found upstream of its own gene.
Determination of the pathway for rhamnose biosynthesis in mycobacteria: cloning, sequencing and expression of the Mycobacterium tuberculosis gene encoding α-D-glucose-1-phosphate thymidylyltransferase
Authors: Ma Yufang , Mills Jonathan A. , Belisle John T. , Vissa Vara , Howell Mark , Bowlin Kelly , Scherman Michael S. , McNeil Michael
Published: June 2009

The mycobacterial cell wall core consists of an outer lipid layer of mycolic acids connected, via arabinogalactan polysaccharide, to an inner peptidoglycan layer. An α-L-rhamnopyranosyl residue has been shown to be a key component linking the mycolated arabinogalactan to the peptidoglycan and, therefore, the biosynthesis of L-rhamnose (Rha) in mycobacteria was investigated as the first step of developing inhibitors of its biosynthesis. Biochemical assays were used to show that dTDP-Rha was synthesized in Mycobacterium smegmatis from α-D-glucose 1-phosphate (α-D-Glc-1-P) and dTTP by the same four enzymic steps used by Escherichia coli and other bacteria. PCR primers based on consensus regions of known sequences of the first enzyme in this series, α-D-Glc-1-P thymidylyltransferase (RfbA) were used to amplify rfbA DNA from M. tuberculosis. The entire rfbA gene was then cloned and sequenced. The deduced amino acid sequence revealed a 31362 Da putative protein product which showed similarity to RfbA proteins of other bacteria (59% identity to that found in E. coli). Sequencing of DNA flanking the rfbA gene did not reveal any of the other rfb genes required for dTDP-Rha biosynthesis. Therefore, the four Rha biosynthetic genes are not clustered in M. tuberculosis. The enzymic activity of the sequenced gene product was confirmed by transformation of E. coli with pBluescript KS(–) containing the rfbA gene from M. tuberculosis. Analysis of enzyme extracts prepared from this transformant revealed an 11-fold increase in α-D-Glc-1-P thymidylyltransferase activity.
Specific and general stress proteins in Bacillus subtilis � a two-dimensional protein electrophoresis study
Authors: Bernhardt J�rg , V�lker Uwe , V�lker Andrea , Antelmann Haike , Schmid Roland , Mach Hiltraut , Hecker Michael
Published: June 2009

A computer-aided analysis of high resolution two-dimensional polyacrylamide gels was used to investigate the changes in the protein synthesis profile in B. subtilis wild-type strains and sigB mutants in response to heat shock, salt and ethanol stress, and glucose or phosphate starvation. The data provided evidence that the induction of at least 42 general stress proteins absolutely required the alternative sigma factor sGB. However, at least seven stress proteins, among them ClpC, ClpP, Sod, AhpC and AhpF, remained stress-inducible in a sigB mutant. Such a detailed analysis also permitted the description of subgroups of general stress proteins which are subject to additional regulatory circuits, indicating a very thorough fine-tuning of this complex response. The relative synthesis rate of the general stress proteins constituted up to 40% of the total protein synthesis of stressed cells and thereby emphasizes the importance of the stress regulon. Besides the induction of these general or rather unspecific stress proteins, the induction of stress-specific proteins is shown and discussed.
Pyruvate carboxylase as an anaplerotic enzyme in Corynebacterium glutamicum
Authors: Peters-Wendisch Petra G. , Wendisch Volker F. , Paul Susanne , Eikmanns Bernhard J. , Sahm Hermann
Published: June 2009

The recent discovery that phosphoenolpyruvate carboxylase (PEPCx) is dispensable for growth and lysine production in Corynebacterium glutamicum implies that this organism possesses (an) alternative anaplerotic enzyme(s). In permeabilized cells of C. glutamicum, we detected pyruvate carboxylase (PCx) activity. This activity was effectively inhibited by low concentrations of ADP, AMP and acetyl-CoA. PCx activity was highest [45 ± 5 nmol min−1 (mg dry wt)−1] in cells grown on lactate or pyruvate, and was about two- to threefold lower when the cells were grown on glucose or acetate, suggesting that formation of PCx is regulated by the carbon source in the growth medium. In cells grown at low concentrations of biotin (< 5 μg I−1), PCx activity was drastically reduced, indicating that the enzyme is a biotin protein. Growth experiments with the wild-type and a defined PEPCx-negative mutant of C. glutamicum on glucose showed that the mutant has a significantly higher demand for biotin than the wild-type, whereas both strains have the same high biotin requirement for growth on lactate and the same low biotin requirement for growth on acetate. These results indicate that (i) PCx is an essential anaplerotic enzyme for growth on glucose in the absence of PEPCx, (ii) PCx is an essential anaplerotic enzyme for growth on lactate even in the presence of PEPCx, and (iii) PCx has no anaplerotic significance for growth on acetate as the carbon source. In support of these conclusions, screening for clones unable to grow on a minimal medium containing lactate, but able to grow on a medium containing glucose or acetate, led to the isolation of PCx-defective mutants of C. glutamicum.
Metabolic flux response to salt-induced stress in the halotoleirant yeast Debaryomyces hansenii
Authors: Neves M. Luisa , Oliveira Rui P. , Lucas Cândida M.
Published: June 2009

The toxic effect of NaCl and KCI on growth of the marine yeast Debaryomyces, hansenii on glucose or glycerol was studied. Above a threshold value, both salts reduced the specific growth rate, specific glucose and glycerol respiration rates and specific glucose fermentation rate, as well as biomass yields. The exponential inhibition constant, k, and minimum toxic concentration, c
min, were similar for all physiological parameters assayed. The effect of either salt on the specific activity of several glycolytic enzymes showed a similar inhibition pattern, although at much lower salt concentrations compared with the physiological parameters. In agreement with published results on glycerol phosphate dehydrogenase stimulation by salt, we present evidence that a general glycolytic flux deviation could occur naturally during salt stress, due to the intrinsic sensitivity of the glycolytic enzymes to intracellular ion concentrations.
Tellurite reductase activity of nitrate reductase is responsible for the basal resistance of Escherichia coli to tellurite
Authors: Avazéri Cécile , Turner Raymond J. , Pommier Jeanine , Weiner Joël H. , Giordano Gérard , Verméglio André
Published: June 2009

Tellurite and selenate reductase activities were identified in extracts of Escherichia coli. These activities were detected on non-denaturing polyacrylamide gels using an in situ methyl viologen activity-staining technique. The activity bands produced from membrane-protein extracts had the same RF values as those of nitrate reductases (NRs) A and Z. Tellurite and selenate reductase activities were absent from membranes obtained from mutants deleted in NRs A and Z. Further evidence of the tellurite and selenate reductase activities of NR was demonstrated using rocket immunoelectrophoresis analysis, where the tellurite and selenate reductase activities corresponded to the precipitation arc of NR. Additionally, hypersensitivity to potassium tellurite was observed under aerobic growth conditions in nar mutants. The tac promoter expression of NR A resulted in elevated tellurite resistance. The data obtained also imply that a minimal threshold level of NR A is required to increase resistance. Under anaerobic growth conditions additional tellurite reductase activity was identified in the soluble fraction on non-denaturing gels. Nitrate reductase mutants were not hypersensitive under anaerobic conditions, possibly due to the presence of this additional reductase activity.
Identification of genes in Rhizobium leguminosarum bv. trifolii whose products are homologues to a family of ATP-binding proteins
Authors: Betzler Michael , Tlolka Inge , Schrempf Hildgund
Published: June 2009

The wild-type Streptomyces lividans 66 genome contains a 4.3 kb amplifiable DNA unit (AUD), and its four ORFs encode proteins that could not be identified by sequence comparison with databases. One of the gene products (encoded by orf-2) was purified and determined to be a novel 23 kDa protein. This protein is synthesized by the wild-type strain, absent in a variant lacking the AUD and overproduced in a variant in which the AUD is amplified (ADS). Immunological studies and analyses by confocal laser microscopy showed that the 23 kDa protein is associated with the substrate hyphae of the wild-type and the ADS-containing variant. Examination by microscopy revealed that the strain carrying the ADS forms bulges within the substrate hyphae and apical vesicles. These bulges have high levels of associated 23 kDa protein and contain storage-like material.
Analysis of the genetic polymorphism between three Streptococcus thermophilus strains by comparing their physical and genetic organization
Authors: Roussel Yvonne , Bourgoin Florence , Guédon Gérard , Pébay Mireille , Decaris Bernard
Published: June 2009

The physical maps of Streptococcus thermophilus CNRZ368 and NST2280 strain: were constructed by analysing PFGE patterns obtained with the low-frequency cutting enzymes SmaI, BssHU and SfiI. Their chromosomes are 1864 and 1840 kb circular molecules, respectively. Comparison of their physical maps with that of the reference A054 strain revealed a relatively conserved organization of the restriction sites. Three variable regions were detected with the map of CNRZ368 whereas 15 were found with the map of NST2280. To construct the genetic maps, probes corresponding to 10 single-copy genes, the rrn genes and the insertion sequences IS1191, IS981 and ISS1 were hybridized to Southern blots of chromosomal DNA digested with the different mapping enzymes. Comparison of the genetic maps of the three strains showed a conserved location of the mapped single-copy genes. However, six rrn loci were present in the chromosome of A054 and CNRZ368 whereas five were present in the NST2280 chromosome. A polymorphism was also found in the copy number of the insertion sequences between the three strains.
Methylosphaera hansonii gen. nov., sp. nov., a psychrophilic, group I methanotroph from Antarctic marine-salinity, meromictic lakes
Authors: Bowman John P. , McCammon Sharee A. , Skerrat Jenny H.
Published: June 2009

Methanotrophic bacteria were enumerated and isolated from the chemocline and surface sediments of marine-salinity Antarctic meromictic lakes located in the Vestfold Hills, Antarctica (68° S 78° E). Most probable number (MPN) analysis indicated that at the chemocline of Ace Lake the methanotroph population made up only a small proportion of the total microbial population and was sharply stratified, with higher populations detected in the surface sediments collected at the edge of Ace Lake and Burton Lake. Methanotrophs were not detected in Pendant Lake. Only a single phenotypic group of methanotrophs was successfully enriched, enumerated and isolated into pure culture from the lake samples. Strains of this group were non-motile, coccoidal in morphology, did not form resting cells, reproduced by constriction, and required seawater for growth. The strains were also psychrophilic, with optimal growth occurring at 10–13°C and maximum growth temperatures of 16–21°C. The ribulose monophosphate pathway but not the serine pathway for incorporation of C1 compounds was detectable in the strains. The guanine plus cytosine (G+C) content of the genomic DNA was 43–46 mol%. Whole-cell fatty acid analysis indicated that 16:1ω8c (37–41%), 16:1ω6c (17–19%), 16:1ω7c (15–19%) and 16:0 (14–15%) were the major fatty acids in the strains. 16S rDNA sequence analysis revealed that the strains form a distinct line of descent in the family Methylococcaceae (group I methanotrophs), with the closest relative being the Louisiana Slope methanotrophic mytilid endosymbiont (91∙8–92∙3% sequence similarity). On the basis of polyphasic taxonomic characteristics the Antarctic lake isolates represent a novel group I methanotrophic genus with the proposed name Methylosphaera hansonii (type strain ACAM 549).
Isolation and characterization of a strong promoter element from the Streptomyces ghanaensis phage I19 using the gentamicin resistance gene (aacC1) of Tn1696 as reporter
Authors: Labes Gabriele , Bibb Mervyn , Wohlleben Wolfgang
Published: June 2009

A promoter-probe shuttle plasmid (pGL7011) containing the promoterless aminoglycoside-O-acetyltransferase I gene (aacC1) of Tn1696 was used to isolate DNA fragments from Streptomyces ghanaensis phage I19 that possessed promoter activity in Streptomyces lividans TK23. Analysis of gentamicin (Gm) resistance levels in Escherichia coli and in S. lividans TK23, and of aacC1 mRNA levels in S. lividans, identified a fragment (F14) that exhibited a high level of promoter activity in both species. Subsequent analysis revealed that the promoter activity of SF14 (a subcloned fragment of F14) was about twice that of ermEp*, one of the strongest characterized actinomycete promoters. SF14 contained two tandemly arranged promoters, 14-1p and p14-llp, with overlapping and adjacent -10 and -35 regions, respectively. Both promoters appear to be recognized with different efficiencies by the major RNA polymerase holoenzyme (Eshrdb) of Streptomyces coelicolor A3(2).
Catabolism of D-glucose by Pseudomonas putida U occurs via extracellular transformation into D-gluconic acid and induction of a specific gluconate transport system
Authors: Schleissner Carmen , Reglero Angel , Luengo José M.
Published: June 2009

Pseudomonas putida U does not degrade D-glucose through the glycolytic pathway but requires (i) its oxidation to D-gluconic acid by a peripherally located constitutive glucose dehydrogenase (insensitive to osmotic shock), (ii) accumulation of D-gluconic acid in the extracellular medium, and (iii) the induction of a specific energy-dependent transport system responsible for the uptake of D-gluconic acid. This uptake system showed maximal rates of transport at 30 ° in 50 mM potassium phosphate buffer, pH 7.0. Under these conditions the Km calculated for D-gluconic acid was 6.7 μM. Furthermore, a different transport system, specific for the uptake of glucose, was also identified. It is active and shows maximal uptake rates at 35 ° in 50 mM potassium phosphate buffer, pH 6.0, with a K
m value of 8.3 μM.
The flgK motility operon of Borrelia burgdorferi is initiated by a s70-like promoter
Authors: Ge Yigong , Old lain G. , Isabelle Saint Girons , Charon Nyles W.
Published: June 2009

A cluster of flagellar genes of Borrelia burgdorferi was identified and sequenced. This cluster comprises an operon, designated the flgK operon, which is initiated by a s70-like promoter. The flgK operon consists of flbF (function unknown), flgK (encoding HAP1), flgL (encoding HAP3) and orfX (function unknown), and maps at 185 kb on the chromosome. In other bacteria, the hook-associated proteins HAP1 and HAP3 connect the flagellar filament to the hook and are required for the last stage of flagellar assembly. Reverse transcriptase-PCR analysis indicated that flbF through to orfX are transcribed as a single mRNA, and primer extension analysis revealed that transcription of the flgK operon is initiated by a s70-like promoter upstream of flbF. Subcloning the flgK promoter element into a promoter probe cat vector revealed that the flgK promoter element had strong activity in both Escherichia coli and Salmonella typhimurium. In addition, when this construct was transformed into a fliA mutant of S. typhimurium which lacked a functional flagellar-specific s28factor, the flgK promoter was still functional. Based on these results, the promoter element of the flagellin gene (fla, hereafter referred to as flaB) was re-examined. flaB encodes the flagellar filament protein, and a sgp33-34-like promoter has been reported to be involved in the transcription of this gene. A transcriptional start point was found 1 bp downstream of the reported start site. The sequence around -10 and -35 are consistent with the presence of a s70-like promoter in addition to the putative sgp33-34-like promoter for flaB. In contrast to the flgK promoter element, no activity was detected after subcloning a flaB promoter element into the promoter probe cat vector. Because a s70-like promoter rather than a unique flagellar sigma factor is involved in the later stage of flagellar assembly, the regulation of B. burgdorferi flagellar genes is evidently different from that of other bacteria.
Molecular and mutational analysis of a DNA region separating two methylotrophy gene clusters in Methylobacterium extorquens AM1
Authors: Chistoserdova Ludmila , Lidstrom Mary E.
Published: June 2009

A region of 14-2 kb has been analysed that is a part of a locus on the Methylobacterium extorquens AM1 chromosome containing a number of genes involved in one-carbon (C1) metabolism, including serine cycle genes, pqq genes, regulatory methanol oxidation genes and the gene for N5,N10-methylene tetrahydrofolate dehydrogenase (mtdA). Fifteen new ORFs have been identified within the new region, and their sequences suggest that they encode the following polypeptides: the C-terminal part of phosphoenolpyruvate carboxylase, malyl-CoA lyase, polypeptides of 9.4 and 31 kDa of unknown function, three putative subunits of an ABC-type transporter, two polypeptides similar to the products of mxaF and mxaJ from M. extorquens AM1 and other methylotrophs, a cytochrome c, three enzymes of folate metabolism, and polypeptides of 13 and 20.5 kDa with no homologues in the protein database. Ten insertion mutations have been generated in the region to determine if the newly identified genes are associated with C1 metabolism. A mutation in mcIA. encoding malyl-CoA lyase, resulted in a C1-minus phenotype, while mutations in the other genes all showed a C1-plus phenotype. It was not possible to obtain null mutants in a putative folate metabolism gene, foIC, implying the necessity of these folate synthesis genes for metabolism of C1 and multicarbon compounds. Mutations in the putative ABC transporter genes, the genes similar to mxaG and mxaJ, and other unidentified ORFs produced double-crossover recombinants with a C1-positive phenotype. Promoter regions have been investigated upstream of orf3 and orf4 using the promoter probe vector pHX200. Transcription from these promoters was weak in wild-type M. extorquens AM1 but increased in regulatory mox mutants.
Extrusion of benzoic acid in
Saccharomyces cerevisiae
by an energy-dependent mechanism
Authors: Henriques Marília , Quintas Célia , Loureiro-Dias Maria C.
Published: June 2009

When grown in the presence of benzoic acid,
Saccharomyces cerevisiae
was able to extrude [14C]benzoic acid when a pulse of glucose was given to preloaded cells. While octanoic, sorbic, hexanoic, salicylic, butyric and propionic acids were also inducers, ethanol and acetic acid were not. The mechanism of extrusion required energy and prior growth in the presence of the inducers. Diethylstilbestrol, an inhibitor of ATPases, prevented benzoic acid extrusion. Propionic acid was not actively extruded in cells adapted to either benzoic or propionic acid, behaving as an appropriate probe to measure intracellular pH. Even though the extrusion mechanism was active, benzoic acid entered the cells by a simple diffusion mechanism.
The thioredoxin reductase system of mycoplasmas
Authors: Ben-Menachem G. , Himmelreich R. , Herrmann R. , Aharonowitz Y. , Rottem S.
Published: June 2009

Representative species of the Mollicutes possess a thioredoxin reductase system (NTS) composed of a low-molecular-mass thioredoxin (TRX) and NADPH-binding thioredoxin reductase (NTR). The TRXs of Mycoplasma pneumoniae and M. capricolum have molecular masses of 11.2 and 12 kDa, respectively, and are stable at 90 °C for 10 min. Both TRXs reacted with monospecific polyclonal antibodies generated against the Bacillus subtilis TRX, but not with anti-Escherichia coli TRX antisera. The M. capricolum and M. pneumoniae NTRs were partially purified and were found to be active with the homologous TRX, but not with the TRX of B. subtilis or E. coli. The NTS activity had an optimal pH of 6.5-7.5 and was dependent on NADPH as an electron donor, a requirement which could not be fulfilled by NADH. The genes encoding the TRX and NTR (trxA and trxB) of M. pneumoniae were cloned and sequenced. The comparative analysis of the predicted amino acid sequence of trxA showed that the 11.2 kDa protein (102 aa) shared 26-68% sequence similarity with products of other known trxA genes and contained the conserved active site Cys-Gly-Pro-Cys. The predicted amino acid sequence of trxB contained 315 residues with a conserved NADPH binding domain and FAD binding domains I and II. The cysteine dithiol redox active region had isoleucine rather than threonine at the active site, as compared with other NTRs. The high activity of the NTS in mycoplasmas suggests that mycoplasmas may have evolved the NTS to protect themselves from the consequences of their self-generated oxidative challenge.
The role of pseudolysogeny in bacteriophage-host interactions in a natural freshwater environment
Authors: Ripp Steven , Miller Robert V.
Published: June 2009

Bacteriophages occur in high numbers in environmental ecosystems and are thus significant mediators of microbial survival and activities. However, interactions between microbial populations and phages in situ have been largely ignored. Current understanding of the process relies on studies performed with well-fed, laboratory-grown host bacteria. The purpose of the experiments reported here was to determine bacteriophage-host interactions under environmentally relevant conditions of nutrient limitation. These studies have revealed the importance of a phenomenon called pseudolysogeny in the maintenance of viral genetic material for extended periods of time in natural ecosystems. Pseudolysogeny is a form of phage-host cell interaction in which the nucleic acid of the phage resides within its starved host in an unstable, inactive state. It is hypothesized that pseudolysogeny occurs due to the cell's highly starved condition. In such cells, there is insufficient energy available for the phage to initiate genetic expression leading to either a true temperate response or to the lytic response. However, upon nutrient addition, the pseudolysogenic state is resolved, resulting in either the establishment of true lysogeny or the initiation of the lytic production of progeny virions. The pseudolysogenic state may explain the long-term survival of viruses in unfavourable environments in which the infective half-life of their virions is relatively short.
Regulation of the TCA cycle and the general amino acid permease by overflow metabolism in Rhizobium leguminosarum
Authors: Walshaw David L. , Wilkinson Adam , Mundy Mathius , Smith Mary , Poole Philip S.
Published: June 2009

Summary: Mutants of Rhizobium leguminosarum were selected that were altered in the uptake activity of the general amino acid permease (Aap). The main class of mutant maps to sucA and sucD, which are part of a gene cluster mdh-sucCDAB, which codes for malate dehydrogenase (mdh), succinyl-CoA synthetase (sucCD) and components of the 2-oxoglutarate dehydrogenase complex (sucAB). Mutation of either sucC or sucD prevents expression of 2-oxoglutarate dehydrogenase (sucAB). Conversely, mutation of sucA or sucB results in much higher levels of succinyl-CoA synthetase and malate dehydrogenase activity. These results suggest that the genes mdh-sucCDAB may constitute an operon. suc mutants, unlike the wild-type, excrete large quantities of glutamate and 2-oxoglutarate. Concomitant with mutation of sucA or sucD, the intracellular concentration of glutamate but not 2-oxoglutarate was highly elevated, suggesting that 2-oxoglutarate normally feeds into the glutamate pool. Elevation of the intracellular glutamate pool appeared to be coupled to glutamate excretion as part of an overflow pathway for regulation of the TCA cycle. Amino acid uptake via the Aap of R. leguminosarum was strongly inhibited in the suc mutants, even though the transcription level of the aap operon was the same as the wild-type. This is consistent with previous observations that the Aap, which influences glutamate excretion in R. leguminosarum, has uptake inhibited when excretion occurs. Another class of mutant impaired in uptake by the Aap is mutated in polyhydroxybutyrate synthase (phaC). Mutants of succinyl-CoA synthetase (sucD) or 2-oxoglutarate dehydrogenase (sucA) form ineffective nodules. However, mutants of aap, which are unable to grow on glutamate as a carbon source in laboratory culture, show wild-type levels of nitrogen fixation. This indicates that glutamate is not an important carbon and energy source in the bacteroid. Instead glutamate synthesis, like polyhydroxybutyrate synthesis, appears to be a sink for carbon and recluctant, formed when the 2-oxoglutarate dehydrogenase complex is blocked. This is in accord with previous observations that bacteroids synthesize high concentrations of glutamate. Overall the data show that the TCA cycle in R. leguminosarum is regulated by amino acid excretion and polyhydroxybutyrate biosynthesis which act as overflow pathways for excess carbon and reductant.
Molecular characterization of the restriction endonuclease gene (scrFIR) associated with the ScrFI restriction/modification system from Lactococcus lactis subsp. cremoris UC503
Authors: Twomey Denis P. , Gabillet Nathalie , Daly Charles , Fitzgerald Gerald F.
Published: June 2009

Summary: The nucleotide sequence of the chromosomally encoded type II ScrFI restriction/modification system from Lactococcus lactis subsp. cremoris UC503 was completed. The ScrFI restriction endonuclease (ENase) has previously been shown to specifically recognize 5’ CCNGG 3’ sites, cleaving after the second cytosine and the degenerate central base. The ENase gene (scrFIR; 862 bp) was located between, and co-directionally transcribed with, two formerly characterized 5-methylcytosine methyltransferase genes, which encode proteins that independently confer protection against ScrFI digestion. scrFIR codes for a protein of 272 amino acids with a predicted molecular mass of 31470 Da, which agrees favourably with a previously estimated molecular mass of 34 kDa for this enzyme. The deduced sequence of this protein did not show any significant homology with known protein sequences, including the isoschizomeric SsoII ENase from ShigeIIa sonnei. The ENase gene was cloned and expressed in Escherichia coli and Lactococcus; however, no in vivo restriction of phage was observed, suggesting that expression of the ENase gene may be repressed, or that the appropriate expression signals may be absent in the cloned constructs. The ability of ScrFI to cleave non-canonically modified 5’ CCNGG 3’ sequences suggested that some ScrFI sites may require complex modifications to fully impair digestion by this enzyme.
Evidence for metabolism of o-xylene by simultaneous ring and methyl group oxidation in a new soil isolate
Authors: Bickerdike S. R. , Holt R. A. , Stephens G. M.
Published: June 2009

Summary: An o-xylene-utilizing Rhodococcus, strain B3, was isolated from enrichments with o-xylene. The pathway for o-xylene degradation was investigated by simultaneous adaptation experiments, studies of product formation by a mutant and fortuitous oxidation studies using trimethylbenzene isomers as substrates. Two pathways were found to operate simultaneously and both were inducible. The first pathway involved the oxidation of a methyl group to form 2-methylbenzyl alcohol, followed by oxidation via the corresponding acid to 3-methylcatechol. The second pathway involved oxidation of the aromatic ring to form a dimethylcatechol. The bulk of the evidence suggests that the initial reaction was catalysed by a monooxygenase rather than a dioxygenase, and that 2,3-dimethylphenol was produced as an intermediate.
Effect of growth rate, nutrient limitation and succinate on expression of TOL pathway enzymes in response to m-xylene in chemostat cultures of Pseudomonas putida (pWW0)
Authors: Duetz Wouter A. , Wind Bea , Kamp Marc , van Andel Johan G.
Published: June 2009

Summary: Previous studies have shown that expression of the toluene and m- and p-xylene degradation pathway in Pseudomonas putida (pWW0) is subject to catabolite repression by succinate. We report here that the expression level of the upper part of this so-called TOL pathway in cells grown in chemostat culture is strongly influenced by nutrient limitation when m-xylene is the sole carbon and energy source. The benzylalcohol dehydrogenase (BADH) levels in cells that are growth-limited by anabolic processes [sulphate (S)-, phosphate (P)- or nitrogen (N)-limiting conditions] were 3-12% of those in cells growing under oxygen limitation (when catabolism limits growth). BADH levels under S-, P- and N-limitation were further decreased (three- to fivefold) when succinate was supplied in addition to m-xylene. Levels of the meta-cleavage pathway enzyme catechol 2,3-dioxygenase were less affected by the growth conditions but the general pattern was similar. Dilution rate also influenced the expression of the TOL pathway: BADH levels gradually decreased with increasing dilution rates, from 1250 mU (mg protein)−1 at D = 0.05 h−1 under m-xylene limitation to 290 mU (mg protein)−1 at D = 0.58 h−1 (non-limited growth). BADH levels were shown to be proportional to the specific affinity whole cells for m-xylene. It may, therefore, be expected that natural degradation rates are adversely affected by anabolic nutrient limitations, especially at relatively low concentrations of the xenobiotic compound.
Generalized transduction in the potato blackleg pathogen Erwinia carotovora subsp. atroseptica by bacteriophage M1
Authors: Toth I. K. , Mulholland V. , Cooper V. , Bentley S. , Shih Y.-L. , Perombelon M. C. M. , Salmond G. P. C.
Published: June 2009

Summary: Using enrichment methods, a new bacteriophage (M1) was isolated, which is capable of generalized transduction in Erwinia carotovora subsp. atroseptica (Eca) strain SCRI1043. M1 is probably a virulent phage and contains double-stranded DNA of approximately 43 kb. Transduction frequencies for a number of chromosomal markers and plasmid pHCP2 were established, and conditions for transduction optimized. UV irradiation of the lysates prior to transduction enhanced the transduction frequency. M1 infected over 25% of Eca strains tested and so may be useful both for the genetic analysis of a number of Eca isolates and for the transductional transfer of selectable markers between strains.
Localization of enzymically enhanced heavy metal accumulation by Citrobacter sp. and metal accumulation in vitro by liposomes containing entrapped enzyme
Authors: Jeong Byeong C. , Hawes Chris , Bonthrone Karen M. , Macaskie Lynne E.
Published: June 2009

Summary: A heavy-metal-accumulating Citrobacter sp. has been used for the treatment of metal-laden industrial wastes. Metal uptake is mediated via a cell-bound phosphatase that liberates inorganic phosphate which precipitates with heavy metals as cell-bound metal phosphate. A phosphatase-deficient mutant accumulated little UO2+
2, while a phosphatase-overproducing mutant accumulated correspondingly more metal, with a uranium loading equivalent to the bacterial dry weight achieved after 6 h exposure of resting cells to uranyl ion in the presence of phosphatase substrate (glycerol 2-phosphate). The phosphatase, visualized by immunogold labelling in the parent and overproducing strains, but not seen in the deficient mutant, was held within the periplasmic space with, in some cells, a higher concentration at the polar regions. Enzyme was also associated with the outer membrane and found extracellularly. Accumulated uranyl phosphate was visible as cell-surface- and polar-localized deposits, identified by energy-dispersive X-ray analysis (EDAX), proton-induced X-ray emission analysis (PIXE) and X-ray diffraction analysis (XRD) as polycrystalline HUO2PO4.4H2O. Nuclaation sites for initiation of biocrystallization were identified at the cytoplasmic and outer membranes, prompting consideration of an in vitro biocatalytic system for metal waste remediation. Phosphatidylcholine-based liposomes with entrapped phosphatase released phosphate comparably to whole cells, as shown by 31P NMR spectroscopy in the presence of ‘IMMR-silent’ 112Cd2+. Application of liposome-immobilized enzyme to the decontamination of uranyl solutions was, however, limited by rapid fouling of the biocatalyst by deposited uranyl phosphate. It is suggested that the architecture of the bacterial cell surface provides a means of access of uranyl ion to the inner and outer membranes and enzymically liberated phosphate in a way that minimizes fouling in whole cells.
Measurement of cytoplasmic pH of the alkaliphile Bacillus lentus C-125 with a fluorescent pH probe
Authors: Aono Rikizo , Ito Masahiro , Horikoshi Koki
Published: June 2009

Summary: A method was established to measure the cytoplasmic pH of the facultative alkaliphilic strain, Bacillus lentus C-125. The bacterium was loaded with a pH-sensitive fluorescent probe, 2′,7′-bis-(2-carboxyethyl)-5 (and -6)-carboxyfluorescein (BCECF), and cytoplasmic pH was determined from the intensity of fluorescence of the intracellular BCECF. The activity of the organism to maintain neutral cytoplasmic pH was assessed by measuring the cytoplasmic pH of the cells exposed to various pH conditions. The cytoplasmic pH maintenance activity of C-125 increased with increasing culture pH, indicating that the activity was regulated in response to the culture pH.
Adhesion of Pseudomonas aeruginosa to silicone rubber in a parallel plate flow chambe in the absence and presence of nutrient broth
Authors: Habash Marc B. , van der Mei Henny C. , Reid Gregor , Busscher Henk J.
Published: June 2009

Summary: The physico-chemical cell-surface properties of Pseudomonas aeruginosa Al and its adhesion to silicone rubber under flow were compared for cells suspended in phosphate-buffered saline (PBS) or PBS supplemented with 2% nutrient broth. Addition of 2% nutrient broth to cells suspended in PBS yields minimal growth and did not significantly change the mean zeta potential of the organisms, which was around -13 mV. However, a comparatively larger proportion of the organisms had more negative zeta potentials in the presen of nutrient broth. This change was concurrent with a slight decrease in cell-surface hydrophobicity, as measured by water contact angles, from 119° to around 112°. The initial deposition rate of P. aeruginosa AK1 to silicone rubber, as studied in a parallel plate flow chamber, increased from 344 cm−2 s−1 in the absence of nutrient broth to 505 cm−2 s−1 in its presence. No stationary level of adhesion was observed in the presence of nutrient broth, instead the number of adhering cells increased steadily at a rate of approximately 85 cm−2 s−1. Fluorescent staining of adhering cells demonstrated that for adhesion from buffer only 2% of the adhering cells were metabolically active, whereas in case of deposition from PBS supplemented with nutrient broth, 67% of the adhering cells were metabolically active. It is concluded that the deposition rates measured in the parallel plate flow chamber with 2% nutrient broth added to the PBS suspension represent an interplay of adhesion and surface-associated growth.
Salmonella typhi mutants defective in anaerobic respiration are impaired in their ability to replicate within epithelial cells
Authors: Contreras Inés , Toro Cecilia S. , Troncoso Gonzalo , Mora Guido C.
Published: June 2009

Summary: By using MudJ (Kan,lac)-directed operon fusion technology, mutants ofSalmonella typhiwhose gene expression is induced under anaerobic growth conditions were isolated. Characterization of their phenotypes and regulatory properties revealed that two of the mutants were unable to use nitrate as a terminal electron acceptor in the absence of oxygen, suggesting that they were defective in nitrate reductase activity. Anaerobic induction of these fusions did not further increase in response to nitrate. Strains carrying an additional mutation inoxrAwere constructed. They showed a lower level of β-galactosidase expression both aerobically and anaerobically; however, the ratios of anaerobic induction remained unaltered. These MudJ insertions mapped to the 17-19 min region of the chromosome. Based upon their phenotypes and mapping, one of the mutants probably possessed amodC (chlD):: MudJ insertion and the other amoaA (chlA):: MudJ insertion. A third mutant was unable to use either nitrate or fumarate as a terminal electron acceptor. All three mutants showed a reduced ability to enter into and proliferate within HEp-2 epithelial cells. TheoxrAmutation enhanced entry and proliferation of both the wild-type cells and the three mutants. Taken together, these results suggest that anaerobic respiration plays a role inS. typhiinvasiveness.
Computer-based analyses of the protein constituents of transport systems catalysing export of complex carbohydrates in bacteria
Authors: Paulsen Ian T. , Beness Alison M. , Saier Milton H.
Published: June 2009

Summary: Bacteria synthesize and secrete an array of complex carbohydrates including exopolysaccharides (EPSs), capsular polysaccharides (CPSs), lipopolysaccharides (LPSs), lipo-oligosaccharides (LOSs) and teichoic acids (TCAs). We have analysed the families of homologous proteins that appear to mediate excretion of complex carbohydrates into or across the bacterial cell envelope. Two principal families of cytoplasmic-membrane transport systems appear to drive polysaccharide export: polysaccharide-specific transport (PST) systems and ATP-binding cassette-2 (ABC-2) systems. We present evidence that the secretion of CPSs and EPSs, but not of LPSs, LOSs or TCAs via a PST or ABC-2 system requires the presence of a cytoplasmic-membrane-periplasmic auxiliary protein (MPA1 or MPA2, respectively) in both Gram-negative and Gram-positive bacteria as well as an outer-membrane auxiliary (OMA) protein in Gram-negative bacteria. While all OMA proteins are included within a single family, MPA1 and MPA2 family proteins are not demonstrably homologous to each other, even though they share common topological features. Moreover, MPA1 family proteins (which function with PST systems), but not MPA2 family proteins (which function with ABC-2 systems), possess cytoplasmic ATP-binding domains that may either exist as separate polypeptide chains (for those from Gram-positive bacteria) or constitute the C-terminal domain of the MPA1 polypeptide chain (for those from Gram-negative bacteria). The sizes, substrate specificities and regions of relative conservation and hydrophobicity are defined allowing functional and structural predictions as well as delineation of family-specific sequence motifs. Each family is characterized phylogenetically.
A Streptomyces griseus gene (sgaA) suppresses the growth disturbance caused by high osmolality and a high concentration of A-factor during early growth
Authors: Ando Noriko , Ueda Kenji , Horinouchi Sueharu
Published: June 2009

Summary: A-factor (2-isocapryloyl-3R-hydroxymethyl-γ-butyrolactone), produced in a growth-dependent manner, switches on secondary metabolite formation and morphological differentiation in Streptomyces griseus, presumably by binding to the A-factor receptor protein (ArpA)-DNA complex and releasing the repression caused by ArpA. In the A-factor-deficient mutant strain S. griseus HH1 a large deletion includes afsA which is required for A-factor production. Growth and aerial mycelium formation of strain HH1 on media containing high concentrations of sucrose, sorbitol, mannitol, KCI or NaCI was disturbed by the presence of a large amount of A-factor supplied either exogenously or by a high-copy-number plasmid carrying afsA. This disturbance did not occur on media of normal osmolality and was observed only when A-factor was supplied during the very early stage of growth, about 8 h after inoculation. In addition, neither the wild-type strain nor S. griseus KM7 defective in ArpA exhibited the disturbance. These observations suggest that the presence of a large amount of A-factor during the very early stage of growth, probably during the A-factor-sensitive stage, triggered abrupt and disordered expression of some genes. The effect was apparently mediated through ArpA in the A-factor regulatory cascade and disturbed the physiology of strain HH1 under high osmolality. A gene that suppressed the disturbance was identified 5.5 kb upstream of the afsA locus in the wild-type strain. The gene, named sgaA, encoded a protein of 264 aa with a calculated molecular mass of 28 kDa.
SpoOA represses transcription of the cry toxin genes in Bacillus thuringiensis
Authors: Poncet Sandrine , Dervyn Etienne , Klier André , Rapoport Georges
Published: June 2009

Summary: The DNA regions upstream from the genes encoding polypeptides of Bacillus thuringiensis subsp. israelensis larvicidal crystals (cry4A, cry4B, cry11A) contain sequences with similarities to the spoOA box of Bacillus subtilis (or ‘OA’ box) and the promoter recognized by the σH-associated RNA polymerase of B. subtilis. Expression of cry-lacZ transcriptional fusions was analysed in various B. thuringiensis genetic backgrounds. The early transcription of the toxin genes was not sporulation-dependent, whereas the late-stage expression at t
4-6 was σE-dependent. Primer extension analysis confirmed that the cry4-and cry11-type toxin genes were weakly transcribed during the transition phase; expression analysis of a cry11A'-lacZ transcriptional fusion in B. subtilis sporulation mutants confirmed the involvement of the σH-RNA polymerase. Primer extension analysis showed that in B. thuringiensis subsp. israelensis, the cry4A and cry11A gene transcription observed at the end of the growth stage was turned off at the beginning of the sporulation phase. The DNA region located upstream from the cry11A gene promoter including the putative ‘OA’ box was deleted. This led to a derepression of the expression of the cry11A operon. These results suggest that the cry4A, cry4B and cry11A toxin genes of B. thuringiensis subsp. israelensis are transcribed during the transition phase by the RNA polymerase associated with the σH factor and are subject to SpoOA repression.
Sequence and analysis of a 31 kb segment of the Bacillus subtilis chromosome in the area of the rrnH and rrnG operons
Authors: Liu Hongtu , Haga Koki , Yasumoto Kenji , Ohashi Yukio , Yoshikawa Hirofumi , Takahashi Hideo
Published: June 2009

Summary: A 31 141 bp continuous nucleotide sequence in the region from trnl to pNEXT52 in the Bacillus subtilis 168 genome was determined. In the region, there were 22 ORFs, two complete rRNA operons, and five tRNA genes. It was deduced that the function of one of the ORFs was similar to that of a sigma factor belonging to the ECF (extra-cytoplasmic functions) subfamily. The gene cluster feuA, B, C reported previously for other strains of B. subtilis was also found in strain 168 and located in this region.