bluuebunny/crossref_metadata_2025 huggingface dataset:
Tyrosine kinase activity in Pseudomonas aeruginosa
Authors: South Suzanne L. , Nichols Robert , Montie Thomas C.
Published: October 2006

Previous evidence showed that b‐ and a‐type flagellins of Pseudomonas aeruginosa are modified in vivo by phosphorylation at tyrosine. This research was designed to demonstrate phosphorylation of flageliin at tyrosine in vitro. Evidence presented showed that flageilin is labelled by [γ‐32P]‐ATP, but not by [α‐32P]‐ATP, when incubated with cell envelope fractions. Results suggested that autophosphoryiation of a 42 kDa membrane protein occurred. No activity was detected in cytoplasmic fractions. Flagellin protein was identified by flagella‐specific monoclonal antibody (mAb) and was labelled with anti‐phosphotyrosine mAb. Confirmation of tyrosine kinase activity was shown by labelling of synthetic poly (Glu:Tyr) as a substrate with [γ‐32P]‐ATP. Labelling of poly (Glu:Tyr) was heat sensitive and time dependent. Labelled phosphotyrosine was observed in partial acid hydrolysates of substrates. Using poly (Glu:Tyr) as substrate, tyrosine kinase activity was shown to be inhibited by sulphydryl reagents. It appears that tyrosine kinase and flagellin phosphorylation occur in several Pseudomonas spp. Location of phosphotyrosine in a conserved region of flagellin may serve as a cell signal so that intact flagellin is appropriately exported.
Targeted mutagenesis of the phospholipase D gene results in decreased virulence ofCorynebacterium pseudotuberculosis
Authors: McNamara Peter J. , Bradley Gregory A. , Songer J. Glenn
Published: October 2006

The chromosomal gene encoding the phospholipase D fromCorynebacterium pseudotuberculosis(biovar ovis) isolate Whetten 1 was replaced with an allele containing a nonsense mutation. The virulence of the mutant strain (W1.31r1) and the isogenic parental strain were then compared by inoculation of goats. The with‐type strain caused abscessation at the site of infection, which then spread to the regional lymph node, while W1.31r1 had a reduced ability to establish a primary infection and was incapable of dissemination. Our results confirm that phospholipase D is a virulence determinant ofC. pseudotuberculosisthat increases the persistence and spread of the bacteria within the host.
Two native plasmids ofPseudomonas syringaepathovar tomato strain PT23 share a large amount of repeated DNA, including replication sequences
Authors: Murillo Jesus , Keen Noel T.
Published: October 2006

Strain PT23 ofPseudomonas syringaepv, tomato contains four native plasmids, designated A, B, C, and D. By DNA hybridization of genomic and plasmid DNA digests from the wild type and a plasmid‐cured strain, we determined thatc. 61 kb (c.74%) of pPT23B is repeated in pPT23A and onlyc.17 kb (c.21%) is in single copy in strain PT23. pPT23B also contains DNA repeated in the chromosome that occurs in three DNA fragments of 0.6, 4.6, and 9.6 kb that might be transposable elements. Additionally, the 9.6 kb fragment also shares sequences with the three other plasmids of strain PT23. By DNA hybridization with the origin of replication from a native plasmid ofP. syringaepv. syringae andin vivoreplication tests, we identified the origins of replication of plasmids A, B, and D and showed that they cross‐hybridize. The putative par region from pPT23 A has also been identified and is not conserved in the other three native plasmids from strain PT23. By using the defined minimal origin of replication from pPT23 A as a probe, we showed that it is highly conserved in 14 strains belonging to nine different pathovars ofP. syringaeand that as many as five different native plasmids with closely related origins of replication coexist in the same cell. The duplication and reorganization of plasmids might therefore occur at high frequency and could be responsible for the existence of large numbers of native plasmids inP. syringaestrains.
The interaction of a recombinant cecropin/melittin hybrid peptide with the outer membrane of Pseudomonas aeruginosa
Authors: Piers K. L. , Hancock R. E. W.
Published: October 2006

A cecropin/melittin hybrid peptide (CEME) produced by recombinant DNA procedures was tested for its ability to interact with the outer membrane of Pseudomonas aeruginosa and found to have identical biological properties to that of chemically synthesized CEME. CEME was shown to kill P. aeruglnosa and permeabilize its outer membrane to lysozyme and 1‐N‐phenylnaphthlyamine, in some cases better than other antimicrobial agents and permeabilizers. CEME demonstrated a high‐binding affinity to purified P. aeruginosa lipopolysaccharide (LPS) and LPS in whole‐cell environments. These data provide information on the molecular mechanism of CEME antimicrobial activity and strongly suggest that it is taken up across the outer membrane by the self‐promoted uptake pathway.
Regulation of acetyl phosphate synthesis and degradation, and the control of flagellar expression in Escherichia coli
Authors: Prüβ Birgtt M. , Wolfe Alan J.
Published: October 2006

We investigated the relationship between Escherichia coli flagellar expression and the regulation of acetyl phosphate synthesis and degradation. Using cells either wild type for acetyl phosphate metabolism or defective for phosphotransacetylase or acetate kinase, or both, we measured flagellar expression and the intracellular concentration of acetyl phosphate relative to growth phase and temperature. Under the conditions tested, we found that elevated levels of acetyl phosphate corresponded to inhibition of flagellar synthesis. To extend these observations, we measured the intracellular concentration of acetyl‐CoA, the level of expression from the pta and ackA promoters, and the activities of phosphotransacetylase and acetate kinase derived from cell lysates. Relative to increasing culture density, acetyl‐CoA levels and expression from both the pta and ackA promoters decreased. Relative to Increasing temperature, expression from the ackA promoter decreased and phosphotransacetylase activity increased. In contrast, temperature had little or no effect on either acetate kinase activity or expression from the pta promoter. We propose that cells regulate intracellular acetyl phosphate concentrations relative to growth phase and temperature by modulating the availability of acetyl‐CoA, the expression of ackA, and the activity of phosphotransacetylase.
Properties of Acinetobacter calcoaceticus recA and its contribution to intracellular gene conversion
Authors: Gregg‐Jolly Leslie A. , Ornston L. Nicholas
Published: October 2006

The Acinetobacter calcoaceticus pcaJ and catJ genes, nearly identical in DNA sequence, differ in transcriptional control and are separated by more than 20 kb of chromosomal DNA. The pcaJ3125 mutation is repaired frequently in organisms containing the wild‐type catJ gene. This high‐frequency repair is eliminated in strains iacking the catJ gene, which suggests that recombination between the homologous catJ and pcaJ genes contributes to the high‐frequency repair of the pcaJ3125 mutation. We report here that the high‐frequency repair also requires a functional recA gene. The A. calcoaceticus recA gene was cloned in Escherichia coli by complementation of a recA mutation in the host strain. The nucleotide sequence of a 1506bp DNA fragment containing A. calcoaceticus recA was determined. The amino acid sequences of RecA from E. coli and A. calcoaceticus shared 71% identity. The DNA sequences differed in that a consensus binding site for binding of LexA repressor, represented upstream from recA in E. coli, is not evident in the corresponding region of the A. calcoaceticus DNA sequence. A Tn5 insertion was introduced into the A. calcoaceticus recA gene. Selection for Tn5‐encoded kanamycin resistance allowed the inactivated recA gene to be recombined by natural transformation into the A. calcoaceticus chromosome. Strains that had acquired the mutant gene were sensitive to both MMS and u.v. light, were deficient in natural transformation, and failed to carry out catJ‐dependent high‐frequency repair of the pcaJ3125 mutation.
Mosaic pbpX genes of major clones of penicillin‐resistant Streptococcus pneumoniae have evolved from pbpX genes of a penicillin‐sensitive Streptococcus oralis
Authors: Sibold Claus , Henrichsen Jørgen , König Andrea , Martin Christiane , Chalkley Lynda , Hakenbeck Regine
Published: October 2006

Penicillin‐resistant clinical isolates of Streptococcus pneumoniae contain mosaic penicillin‐binding protein (PBP) genes that encode PBPs with decreased affinity for β‐lactam antibiotics. The mosaic blocks are believed to be the result of gene transfer of homologous PBP genes from related penicillin‐resistant species. We have now identified a gene homologous to the pneumococcal PBP2x gene (pbpX) in a penicillin‐sensitive Streptococcus oralis isolate M3 from South Africa that diverged by almost 20% from pbpX of penicillin‐sensitive pneumococci, and a central sequence block of a mosaic pbpX gene of Streptococcus mitis strain NCTC 10712. In contrast, it differed by only 2‐4% of the 1 to 1.5 kb mosaic block in pbpX genes of three genetically unrelated penicillin‐resistant S. pneumoniae isolates, two of them representing clones of serotype 6B and 23F, which are prevalent in Spain and are also already found in other countries. With low concentrations of cefotaxime, transformants of the sensitive S. pneumoniae R6 strain could be selected containing pbpX genes from either S. mitis NCTC 10712 or S. oralis M3, demonstrating that genetic exchange can already occur between β‐lactam‐sensitive species. These data are in agreement with the assumption that PBPs as penicillin‐resistance determinants have evolved by the accumulation of point mutations in genes of sensitive commensal species.
Prokaryotic HU and eukaryotic HMG1: a kinked relationship
Authors: Bianchi Marco E.
Published: October 2006

HU and IHF proteins have long been considered the prokaryotic analogues of eukaryotic histones. Their ability to bend DNA, however, is distinctly similar to that of eukaryotic HMG‐box proteins, a recently identified family of chromatin components and transcription factors. In some conditions, HU and HMG1‐like proteins can even be swapped, both in vitro and in vivo. In spite of this, HU/IHF and HMG‐box proteins are not evolutionarily related, and represent two independent solutions for the same biochemical problem.
Altered pH lysine signalling mutants of cadC, a gene encoding a membrane‐bound transcriptional activator of the Escherichia coli cadBA operon
Authors: Dell Cheryl L. , Neely Melody N. , Olson Eric R.
Published: October 2006

The Escherichia coli CadC protein is required for activation of cadBA transcription under conditions of low external pH and exogenous lysine. cadBA encodes proteins involved in the decarboxylation of lysine to cadaverine, and cadaverine excretion. Sequence analysis suggested that CadC contains a single transmembrane segment separating a DNA‐binding domain in the amino terminus from a periplasmic domain. Western analysis of subcellular fractions demonstrated that CadC is expressed and concentrated in the cytoplasmic membrane in cells grown either at an inducing pH (pH5.8) or at a non‐inducing pH (pH7.6.). Eight cadC mutants were isolated based on their ability to confer expression of a cadA‐lacZ fusion independent of low external pH or exogenous lysine. Five of these mutants expressed the cadA‐lacZ fusion at both pH 5.8 and pH 7.6, but retained the requirement for the lysine signal while the other three mutants displayed pH independence in the presence of lysine, and lysine independence at pH 5.8 but not at pH 7.6. These results support a model in which CadC is a membrane‐bound transcriptional activator of the cadBA operon capable of sensing (directly or indirectly) signals generated outside the cytoplasmic membrane as a consequence of acidic pH and lysine.
Information essential for cell‐cycle‐dependent secretion of the 591‐residue Caulobacter hook protein is confined to a 21‐amino‐acid sequence near the N‐terminus
Authors: Kornacker M. G. , Newton A.
Published: October 2006

Recent findings suggest that axial flagellar proteins and virulence proteins of Gram‐negative bacteria are exported from the cytoplasm via conserved trans‐location systems. To identify residues essential for secretion of flagellar axial proteins we examined the 591‐residue Caulobacter crescentus flagellar hook protein. Western blot assays of the culture media of strains producing mutant hook proteins show that only residues 38–58 are essential for its secretion to the cell surface. We discuss the observation that this unprocessed 21‐residue sequence is not conserved in other axial proteins and does not correspond to the SGL‐, ANN LAN‐ and heptad repeat motifs that are located Just upstream of the essential secretion information in the hook protein and are conserved near the N‐termini of other axial proteins. These motifs, for which an essential role in export or assembly has been proposed, are required for motility. However, we also demonstrate that hook protein can only be secreted when the flagellar basal body is present in the cell envelope. The cell‐cycle regulation of hook protein secretion confirms the specificity of the assay used in these studies and suggests that the basal body itself may serve as a secretion channel for the hook protein.
Expression of seven members of the gene family encoding secretory aspartyl proteinases in Candida albicans
Authors: Hube B. , Monod M. , Schofield D. A. , Brown A. J. P. , Gow N. A. R.
Published: October 2006

The opportunistic fungal pathogen Candida albicans produces secretory aspartyl proteinases, which are believed to be virulence factors in infection. We have studied the in vitro expression of seven known members of the SAP gene family in a range of strains and serotypes by Northern analysis. SAP1 and SAP3 were regulated during phenotypic switching between the white and opaque forms of the organism. The SAP2 mRNA, which was the dominant transcript in the yeast form, was found to be autoinduced by peptide products of Sap2 activity and to be repressed by amino acids. The expression of the closely related SAP4‐SAP6 genes was observed only at neutral pH during serum‐induced yeast to hyphal transition. No SAP7 mRNA was detected under any of the conditions or in any of the strains tested. Our data suggest that the various members of the SAP gene family may have distinct roles in the colonization and invasion of the host.
Proteolytic cleavage and cell wall anchoring at the LPXTG motif of surface proteins in Gram‐positive bacteria
Authors: Navarre William Wiley , Schneewind Olaf
Published: October 2006

Many surface proteins are thought to be anchored to the cell wall of Gram‐positive bacteria via their C‐terminus. Cell wall anchoring requires a specific sorting signal, normally located at the predicted C‐terminus of surface proteins. Here we show that when placed into the middle of a polypeptide chain, the sorting signal causes the specific cleavage of the precursor as well as the cell wall anchoring of its N‐ terminal fragment, while the C‐terminal fragment remains within the cytoplasm. N‐terminal sequencing of the C‐terminal cleavage fragment suggests that the cleavage site is located between threonine (T) and glycine (G) of the LPXTG motif, the signature sequence of cell wall sorting signals. All surface proteins harbouring an LPXTG sequence motif may therefore be cleaved and anchored by a universal mechanism. We also propose a novel hypothesis for the cell wall linkage of surface proteins in Gram‐positive bacteria.
Escherichia coli tyrT gene transcription is sensitive to DNA supercoiling in its native chromosomal context: effect of DNA topoisomerase IV overexpression on tyrT promoter function
Authors: Free Andrew , Dorman Charles J.
Published: October 2006

We have investigated the in vivo DNA supercoiling sensitivity of the Escherichia coli tRNAtyr1 gene (tyrT) promoter in its normal chromosomal location. Here, the native tyrT promoter is found to be exquisitely sensitive to mutations and to drugs which alter the level of DNA supercoiling. We show that the response of the tyrT promoter to supercoiling is qualitatively similar to that of a known supercoiling‐sensitive tRNA gene promoter, hisR. Specifically, treatments which increase in vivo DNA supercoiling levels enhance transcription of these tRNA genes. Particularly striking is the strong enhancement of expression from both promoters by a transposon insertion mutation in the topA gene encoding DNA topoisomerase i. This phenotypic effect can be complemented by providing active topoisomerase I in trans from a recombinant plasmid. Interestingly, it can also be complemented by overexpression of the genes encoding the subunits of DNA topoisomerase IV. We believe that this is the first demonstration that DNA topoisomerase IV can influence gene expression and it suggests that DNA topoisomerase I is partially redundant, at least in E. coli.
Repeated polyketide synthase modules involved in the biosynthesis of a heptaene macrolide by Streptomyces sp. FR‐008
Authors: Hu Zhihao , Bao Kai , Zhou Xiufen , Zhou Qi , Hopwood David A. , Kieser Tobias , Deng Zixln
Published: October 2006

Genes for biosynthesis of a Streptomyces sp. FR‐008 heptaene macrolide antibiotic with antifungal and mosquito larvicidal activity were cloned in Escherichia coli using heterologous DNA probes. The cloned genes were implicated in heptaene biosynthiesis by gene replacement. The FR‐008 antibiotic contains a 38‐membered, poiyketide‐derived macrolide ring. Southern hybridization using probes encoding domains of the type i modular erythromycin polyketide synthase (PKS) showed that the Streptomyces sp. FR‐008 PKS gene cluster contains repeated sequences spanning c. 105 kb of contiguous DNA; assuming c. 5 kb for each PKS module, this is in striking agreement with the expectation for the 21‐step condensation process required for synthesis of the FR‐008 carbon chain. The methods developed for transformation and gene replacement in Streptomyces sp. FR‐008 make it possible to genetically manipulate polyene macrolide production, and may later lead to the biosynthesis of novel polyene macrolides.
Iron, DtxR, and the regulation of diphtheria toxin expression
Authors: Tao Xu , Schiering Nikolaus , Zeng Hui‐yan , Ringe Dagmar , Murphy John R.
Published: October 2006

In recent years considerable advances have been made in the understanding of the molecular basis of iron‐mediated regulation of diphtheria toxin expression. The tox gene has been shown to be regulated by the heavy metal ion‐activated regulatory element DtxR. In the presence of divalent heavy metal ions, DtxR becomes activated and binds to a 9 bp interrupted palindromic sequence. The consensus‐binding site has been determined by both the sequence analysis of DtxR‐responsive operators cloned from genomic libraries of Corynebacterium diphtheriae as well as by in vitro genetic methods using cyclic amplification of selected targets (CAST‐ing). it is now clear that DtxR functions as a global iron‐sensitive regulatory element in the control of gene expression in C. diphtheriae. In addition, the metal ion‐activation domain of DtxR is being characterized by both mutational analysis and determination of the X‐ray structure at 3.0 Å resolution.
Building bridges: disulphide bond formation in the cell
Authors: Bardwell James C. A.
Published: October 2006

Disulphides are often vital for the folding and stability of proteins. Dedicated enzymatic systems have been discovered that catalyse the formation of disulphides in the periplasm of prokaryotes. These discoveries provide compelling evidence for the actual catalysis of protein foldingin vivo.Disulphide bond formation inEscherichia coliis catalysed by at least three ‘Dsb’ proteins; DsbA, ‐B and ‐C. The DsbA protein has an extremely reactive, oxidizing disulphide which it simply donates directly to other proteins. DsbB is required for the reoxidation of DsbA. DsbC is active in disulphide rearrangements and appears to work synergistically with DsbA. The relative rarity of disulphides in cytoplasmic proteins appears to be dependent upon a disulphide‐destruction machine. One pivotal cog in this machine is thioredoxin reductase.
The RTX haemolysins ApxI and ApxII are major virulence factors of the swine pathogenActinobacillus pleuropneumoniae:evidence from mutational analysis
Authors: Tascón Rubén I. , Vázquez‐Boland José A. , Gutiérrez‐Martín César B. , Rodríguez‐Barbosa Ignacio , Rodríguez‐Ferri ElZías F.
Published: October 2006

The involvement of the RTX haemolysins (Apxl and ApxII) of the swine pathogenActinobacillus pleuropneumoniae in virulencewas investigated using haemolysin‐deficient mutants constructed by a mini‐Tn10mutagenesis procedure. Two types of haemolysin mutant with single insertions of the transposon were obtained from a serotype 1 strain producing both ApxI and ApxII. One presented a complete loss of haemolytic activity because of the absence of ApxI and ApxII production. The other displayed weaker haemolysis than the wild type and produced only ApxII. The chromosomal regions flanking mini‐Tn10were cloned and sequenced. In the non‐haemolytic mutant, the transposon had inserted inapxIB, a gene involved in the exportation of ApxI and ApxII toxins. The weakly haemolytic mutant resulted from the disruption of the structural gene for ApxI. Both mutations In theapxIoperon were associated with a significant loss of virulence for mice and pigs, demonstrating that haemolysins are involved inA. pleuropneumoniaepathogenicity. The non‐haemolytic mutant was apathogenic and the weakly haemolytic mutant retained some virulence for pigs, suggesting that both ApxI and ApxII are needed for full virulence.
Identification and characterization of an intervening sequence within the 23S ribosomal RNA genes of Campylobacter jejuni
Authors: Konkel Michael E. , Marconi Richard T. , Mead David J. , Cieplak Witold
Published: October 2006

Campylobacter jejuni is a significant cause of bacterial enteritis in humans. Three of seven C. jejuni isolates examined were found to contain fragmented 23S rRNA. The occurrence of fragmented 23S rRNA correlated with the presence of an intervening sequence (IVS) within the 23S rRNA genes. The IVS is 157 nucleotides in length and replaces an eight nucleotide sequence in the 23S rRNA genes of C. jejuni isolates that contain intact 23S rRNA. The two ends of the IVS share 31 bases of complementarity that could form a stem‐loop structure. Fragmentation of the 23S ribosomal RNA results from the excision of the IVS from the transcribed RNA; the 3’ cleavage site maps within the putative stem‐loop formed by the IVS. Southern hybridization analysis revealed that the IVS is not present in the genomes of isolates that contain intact 23S rRNA, suggesting that the IVS is not derived from Campylobacter chromosomal sequences. The C. jejuni IVS is located at a position analogous to that of the IVSs found in both Salmonella and Yersinia spp.
Proximal and distal sites bind LuxR independently and activate expression of the Vibrio harveyi lux operon
Authors: Miyamoto Carol M. , Smith Eric E. , Swartzman Eiana , Cao Jie‐Gang , Graham Angus F. , Meighen Edward A.
Published: October 2006

The LuxR regulatory protein of Vibrio harveyi as well as the autoinducer molecule, N‐(3‐hydroxybutanoyl) homoserine lactone, are known to be required for expression of luminescence. Although LuxR has been implicated in the activation of the promoter of the lux operon of V. harveyi, and can bind to two distinct sites upstream of the transcription initiation start site, its mode of action is unknown, in the present experiments, mobility shift assays were used to demonstrate that LuxR bound to the distal and proximal sites in an independent rather than co‐operative interaction with a much tighter binding to the distal site. Deletion and mutation analyses of DNA upstream of the lux promoter followed by transconjugation Into V. harveyi in trans using the chloramphenicol acetyl‐transferase (cat) gene as a reporter demonstrated, however, that the proximal site for LuxR was absolutely critical for promoter activation while the distal LuxR site was only necessary for maximum activation. This result was confirmed by mutation of the proximal site which blocked activation of the lux promoter and binding of LuxR to this site, but did not prevent LuxR binding to the distal site.
Characterization of trimethoprim‐ and sulphisoxazole‐resistant Chlamydia trachomatis
Authors: Wang Ling Ling , Henson Elizabeth , McClarty Grant
Published: October 2006

Trimethoprim and sulphisoxazole were used as selective agents in culture to isolate, by a stepwise procedure, a series of Chlamydia trachomatis L2 populations resistant to the cytotoxic effects of the drugs. Two trimethoprim‐resistant populations. L2TriR‐60 and L2TriR‐100, and one sulphonamide‐resistant population. L2SulfR‐100, were characterized in more detail. In addition to being resistant to trimethoprim, L2TriR‐100 was cross‐resistant to metho‐trexate, sensitive to sulphisoxazole and displayed a ribonucleotide auxotrophy similar to that of its parental wild type, C. trachomatis L2. Surprisingly, L2TriR‐100 and L2SulfR‐100 appeared phenotypically identical. Both mutants were highly resistant to trimethoprim, sulphisoxazole, and methotrexate. In contrast to wild‐type C. trachomatis L2, these populations were sensitive to 5‐fluorouracil L2TriR‐100 and L2SulfR‐100 were incapable of taking pyrimidine ribonucleotides from the host cell and no longer synthesized thymidine nucleotides de novo. The pyrimidine requirement of these mutants was met by salvaging host‐cell uracil and thymidine, a property which can account for their drug‐resistance characteristics. L2TriR‐100 and L2Sulfn‐100 could also salvage adenine and guanine. Using L2TriR‐100 as a starting stock, a mutant population resistant to the cytotoxic effects of trimethoprim and 5‐fluorouracil (L2Tri/5‐FU) was selected. L2TFi/5‐FU was resistant to 5‐fluoro‐uracil because it had regained the capacity to take pyrimidine ribonucleotides from the host cell.
Analysis of the rnc locus of Coxiella burnetii
Authors: Zuber Mohammed , Hoover Timothy A. , Powell Bradford S. , Court Donald L.
Published: October 2006

A 3.2 kb EcoRI genomic DNA fragment of Coxiella burnetii was isolated by virtue of Its ability to suppress mucoidy in Eschertchia coli. Nucleotide sequence analysis revealed the presence of the genes homologous to rnc, era and recO of E. coli. Suppression of capsule synthesis, measured by β‐galactosidase expression in Ion cps‐lac fusion strains of E. coli, is caused by gene‐dosage effects of the plasmid‐borne rnc genes of either C. burnetii or E. coli. The rnc gene of C. burnetii complemented rn– E. coli hosts for lambda plaque morphology and stimulation of lambda N gene expression. We also demonstrated heterologous complementation of an E coli strain defective for the expression of Era, an essential protein in E. coli, using the plasmid‐borne C. burnetii era. Under the control of the bacteriophage lambda PL promoter, this 3.2 kb EcoRI DNA fragment directed the synthesis in E. coli of three proteins with approximate molecular masses of 35,27 and 25 kDa. Antibodies against purified E. coli Era protein cross‐reacted with the 35 kDa protein of C. burnetii on Western blots.
Strain variation in the katG region of Mycobacterium tuberculosis
Authors: Zhang Ying , Young Douglas
Published: October 2006

Southern blot analysis of chromosomal DNA from clinical isolates of Mycobacterium tuberculosis using cosmid DNA probes revealed extensive strain variation in the katG region of the genome. In addition to deletion of the katG gene itself in some isoniazid‐resistant strains, adjacent DNA fragments were missing or altered in a range of drug‐sensitive and drug‐resistant isolates. A species‐specific 2 kb Kpnl fragment located 10 kb upstream of katG in M. tuberculosis H37Rv hybridized to fragments of differing size in different clinical isolates and was characterized in detail. Sequence analysis of this fragment showed that it comprised three tandem copies of a novel 75 bp repeat element flanked by multiple copies of the previously described 10 bp major polymorphic tandem repeat of M. tuberculosis (MPTR). The copy number of the 75 bp repeat was found to vary between strains, allowing application of a poly‐merase chain reaction amplification strategy for strain differentiation. These results indicate that the katG region of the M. tuberculosis genome is highly variable and unstable. The presence of repetitive sequences may contribute to instability in this region of the genome.
The tail of a chaperonin: the C‐terminal region of Esctierichia coli GroEL protein
Authors: McLennan Neil F. , McAteer Sean , Masters Millicent
Published: October 2006

The active form of the KSP60 molecular chaperone of Escherichia coli, GroEL, is a pair of seven‐membered rings. We have used site‐directed mutagenesis to construct forms of the 547‐amino‐acid monomer truncated at the C‐terminus. We show here that forms that are 520 amino acids long or longer are close to being fully functional. Removing one further amino acid, however, results in a protein, GroEL519, which retains little function. This truncated form is metabolically stable but is not recovered from the cell in particle form. When synthesized at high levels, it prevents the normal assembly of GroEL547 present in the same cell. When synthesized at low levels, it can be included, probably at low molar ratios, in particles formed by assembly‐competent forms of GroEL. This can be seen as partial complementation of the temperature‐sensitive mutant groEL44. We conclude that amino acid 520 is cruical for particle assembly. GroEL516 has in vivo properties similar to those of GroEL519, but the still shorter form, GroEL504, appears to be inactive.
Neisseria gonorrhoeae acquires mutations in analogous regions of gyrA and parC in fluoroquinolone‐resistant isolates
Authors: Belland R. J. , Morrison S. G. , Ison C. , Huang W. M.
Published: October 2006

Neisseria gonorrhoeae homologues of gyrA and parC have been identified using hybridization probes generated from conserved regions of diverse gyrA genes. These genes have been tentatively identified as gyrA and parC, based on predicted amino acid sequence homologies to known GyrA homologues from numerous bacterial species and to ParC from Escherichia coli and Salmonella typhimurium. The gyrA gene maps to a physical location distant from the gyrB locus on the gonococcal chromosome, which is similar to the situation found in E. coli. The parC gene is not closely linked (i.e. greater than 9 kb) to an identifiable parE gene in N. gonorrhoeae. The gonococcal GyrA is slightly larger than its E. coli homologue and contains several small insertions near the O‐terminus of the predicted open reading frame. A series of ciprofloxacin‐resistant mutants were selected by passage of N. gonorrhoeae on increasing concentrations of the antibiotic. Sequential passage resulted in the selection of isolates with minimum inhibitory concentrations approximately 10000‐fold higher than the parental strain. Mutations within gyrA resulted in low to moderate levels of resistance, while strains with high‐level resistance acquired analogous mutations in both gyrA and parC. Resistance mutations were readily transferred between N. gonorrhoeae strains by transformation. The frequencies of transformation, resulting in different levels of ciprofloxacin resistance, further support the notion that both gyrA and parC genes are invoived in the establishment of extreme levels of ciprofloxacin resistance.
Elucidation of the mechanism of CryIIIA overproduction in a mutagenized strain of Bacillus thuringiensis var. tenebrionis
Authors: Adams Lee F. , Mathewes Shannon , O'Hara Patrick , Petersen Annette , Gürtler Hanne
Published: October 2006

NB176 is a Bacillus thuringiensis mutant derived by λ‐irradiation of NB125 Bacillus thuringiensis var. tenebrionis (Krieg). It exhibits two interesting phenotypes: (i) oligosporogeny and (ii) twofold to threefold overproduction of the CryIIIA protein. Southern profiles of the NB176 strain showed an additional copy(s) of the cryIIIA gene located on a 4 kb HindIII fragment, in addition to the expected cryIIIA gene on a 3 kb HindIII fragment. Each cryIIIA gene‐bearing HindIII fragment was cloned from NB176. The restriction map of the 3 kb HindIII fragment was identical to that published by Donovan and coworkers. Sequencing of the 4 kb HindIII fragment showed no alterations in the promoter region of the cryIIIA gene but did show replacement of the region immediately following the cryIIIA open reading frame with a sequence encoding a transposase with 50% amino acid homology to that of Tn 1000. These findings suggest that the overproduction phenotype of NB176 results from extra copies of the cryIIIA gene produced from a transposition event(s) induced or stabilized by γ‐irradiation. Integration of additional copies of the cryIIIA gene into the native 90MDa plasmid of the wild‐type B. thuringiensis var. tenebrionis strain resulted in strains that made enormous crystals, many possessing greatly enhanced insecticidal activity.
Non‐congruent relationships between variation in emm gene sequences and the population genetic structure of group A streptococci
Authors: Whatmore Adrian M. , Kapur Vivek , Sullivan Derek J. , Musser James M. , Kehoe Michael A.
Published: October 2006

To examine the molecular population genetics of the M protein family of Streptococcus pyogenes (group A Streptococcus), the 5′ regions of polymerase chain reaction‐amplified emm products from 79 M serotypes were sequenced and the phylogeny was compared to estimates of overall genetic relationships among strains determined by multilocus enzyme electrophoresis. Although the 5′emm sequences from several strains designated as distinct M types were identical or almost identical, the overall pattern is characterized by very extensive variation. The composition of distinct emm sequence clusters generally parallels the ability of strains to express serum opacity factor and in some cases historical associations of certain M types with acute rheumatic fever, but not with M types classified as nephritogenic. For many strains there is a lack of congruency between variation in 5′emm sequences and estimates of overall chromosomal relationships, which is undoubtedly due to horizontal transfer and recombination of emm sequences. The results of these studies provide insights into the nature and extent of emm sequence variation and describe how this variation ‘maps’ onto the population genetic structure of extant S. pyogenes lineages. The complexity of emm sequence and streptococcal cell lineage relationships revealed by this analysis has significant implications for understanding evolutionary events generating strain diversity and the epidemiology of S. pyogenes diseases.
NarL‐phosphate must bind to multiple upstream sites to activate transcription the narG promoter of Escherichia coli
Authors: Walker M. S. , DeMoss J. A.
Published: October 2006

The stimulation of Fnr‐dependent transcription from the narG promoter by NarL‐phosphate is known to require a cis‐acting sequence, the NarL box, located approximately 195 bp upstream from the transcription start site, and the interaction of integration host factor (IHF) with a binding site in the intervening region (positions −110 to −140) between the NarL box and the transcription start site. By gel retardation and DNase I protection studies, we have demonstrated that NarL‐phosphate, produced by the reaction of purified NarL with acetyl phosphate, specifically binds to a fragment derived from the upstream region of the narG promoter. The fragment was protected by NarL‐phosphate binding to two distinct regions. One was an extended sequence of approximately 40 bp surrounding the NarL box at −195; the second was located downstream from the IHF‐binding region and included a sequence extending from positions −80 to −120. Alteration by site‐directed mutagenesis of a putative inverted NarL box sequence identified within the downstream protected region in a plasmid containing a narG‐lacZ fusion eliminated the NarL‐phosphate‐mediated stimulation of transcription. NarL‐phosphate bound to the two regions independently from IHF binding and it bound to each site independently when the two sites were separated by cleavage of the promoter fragment. Stimulation of transcription from the narG promoter by NarL‐phosphate appears to result from the formation of a folded protein‐DNA structure created by the binding of NarL‐phosphate to multiple sites on either side of an IHF‐induced bend in the upstream region of the promoter.
afsR2: a previously undetected gene encoding a 63‐amino‐acid protein that stimulates antibiotic production in Streptomyces lividans
Authors: Vögtil Martin , Chang Poa‐Chun , Cohen Stanley N.
Published: October 2006

Earlier work has shown that the afsR genetic locus promotes formation of the pigmented antibiotics actinorhadin and undecylprodiglosin in Streptomyces lividans and its close relative, Streptomyces coeiicolor. A protein designated as AfsR has been implicated in this activity. We report here the existence of a previously unknown gene, afsR2, which is separate from and adjacent to the AfsR‐encoding sequence and which, when present at high copy number, (i) stimulates transcription of biosynthetic and regulatory genes in the actinorhodin gene cluster (act), and (ii) stimulates the synthesis of undecyiprodigiosin., We show that the effects of afsR2 on actinorhodin synthesis are mediated through transcription of the actll‐0RF4 locus, which encodes a transcriptional activator of other genes in the act cluster. Analysis of the oloned afsR2 gene indicates that its activity is the result of the 63‐amino‐acid protein it specifies.
Common ancestry between IncN conjugal transfer genes and macromolecular export systems of plant and animal pathogens
Authors: Pohlman Robert F. , Genetti Heather D. , Winans Stephen C.
Published: October 2006

The DNA sequence of a cluster of pKM101 conjugal transfer genes was determined and aligned with the genetic map of the plasmid. Eighteen genes were identified, at least eight and probably 11 of which are required for efficient conjugation. These tra genes are homologous to and colinear with genes found in the virB operon of Agrobacterium tumefaciens TI plasmids. Seven pKM101 tra genes are also homologous to ptl genes of Bordetella pertussis, which direct the export of pertussis toxin. We used TnphoA to construct translational fusions between pKM101 genes and the Escherichia coli phoA gene, which encodes alkaline phosphatase, and provide evidence that at least 11 of the 18 genes are either fully or partially exported from the cytoplasm.
A protein complex mediating mRNA degradation in Escherichia coli
Authors: Py Béatrice , Causton Helen , Mudd Elisabeth A. , Higgins Christopher F.
Published: October 2006

mRNA degradation in Escherichia coli is mediated by a combination of exo‐ and endoribolucleases. We present evidence for a multiprotein complex which includes at least two enzymes that play important roles in mRNA degradation: the exoribonuclease poly‐nucleotide phosphorylase (PNPase) and the endorlbo‐nuclease RNase E. An activity which impedes the processive activity of PNPase at stem‐loop structures also appears to be associated with the complex. This complex is estimated to have a molecular mass of about 500 kDa and includes several additional poly‐peptides whose functions are unknown. The identification of a complex which includes several activities associated with mRNA degradation has implications for the mechanisms and co‐ordinated control of mRNA degradation.
Cell‐surface‐associated polypeptides CshA and CshB of high molecular mass are colonization determinants in the oral bacterium Streptococcus gordonii
Authors: McNab R. , Jenkinson H. F. , Loach D. M. , Tannock G. W.
Published: October 2006

The human oral bacterium Streptococcus gordonii expresses, on the cell surface, two antigenically related high‐molecular‐mass polypeptides denoted CshA and CshB, encoded by genes at separate chromosomal loci. The precursor form of CshA is composed of four distinct segments: (i) a 41‐amino‐acid residue leader peptide, (ii) W‐terminal 42–878 residues, (iii) residues 879–2417 comprising 13 repeat blocks of 101 amino acid residues and three shorter blocks, and (iv) a C‐terminal anchor domain similar to those present in some other Gram‐positive bacterial cell‐wall polypeptides. Insertional mutations within cshA reduced both cell‐surface hydrophobicity and ability to adhere to oral Actinomyces naeslundii. Insertional mutations in cshB had less effect on hydrophobicity and coadherence. However, expression of both polypeptides was found to be necessary for streptococci to colonize the murine oral cavity.
The Legionella pneumophila icm locus: a set of genes required for intracellular multiplication in human macrophages
Authors: Brand Bettina C. , Sadosky Alesia B. , Shuman Howard A.
Published: October 2006

Legionella pneumophila, the causative agent of Legionnaires’ disease and related pneumonias, infects, replicates within and eventually kills human macrophages. A key feature of the intracellular lifestyle is the ability of the organism to replicate within a specialized phagosome which does not fuse with Iysosomes or acidify. Avirulent mutants that are defective in intracellular multiplication and host‐cell killing are unable to prevent phagosome–Iysosome fusion. In a previous study, a 12kb fragment of the L. pneumophila genome containing the icm locus (intracellular multiplication) was found to enable the mutant bacteria to prevent phagosome‐Iysosome fusion, to multiply intracellularly and to kill human macrophages. The complemented mutant also regained the ability to produce lethal pneumonia in guinea‐pigs. In order to gain information about how L. pneumophila prevents phagosome‐Iysosome fusion and alters other intracellular events, we have studied the region containing the icm locus. This locus contains four genes, icmWXYZ, which appear to be transcribed from a single promoter to produce a 2.1–2.4kb mRNA. The deduced amino acid sequences of the Icm proteins do not exhibit significant similarity to other proteins of known sequence, suggesting that they may carry out novel functions. The icmX gene encodes a product with an apparent signal sequence suggesting that it is a secreted protein. The icmWXYZ genes are located adjacent to and on the opposite strand from the dot gene, which is also required for intracellular multiplication and the ability of L. pneumophila to modify organelle traffic in human macrophages. Five L. pneumophila Icm mutants that had been generated with transposon Tn903dIIlacZ were found to have Inserted the transposon within the icmX, icmY, icmZ and dot genes, confirming their role in the ability of the organism to multiply intracellularly.
Altered intracellular targeting properties associated with mutations in the Legionella pneumophila dotA gene
Authors: Berger Karen H. , Merriam Jennifer J. , Isberg Ralph R.
Published: October 2006

Legionella pneumophila dot mutations cause defects in intracellular targeting of the microorganism within cultured macrophages. Each of the previously characterized dot mutations was shown to be complemented by a single open reading frame designated dotA. The defects caused by the mutations appear to be due to disrupted function of the predicted 1048‐amino‐acid residue DotA protein, and not by polarity effects on a downstream gene. Complementation studies indicated that the product of the dotA53 mutation results in a partially functional DotA protein, consistent with a stable N‐terminal fragment having biological activity.
Isolation of motile and non‐motile insertional mutants of Campylobacter jejuni: the role of motility in adherence and invasion of eukaryotic cells
Authors: Yao Ruijin , Burr Don H. , Doig Peter , Trust Trevor J. , Niu Haiying , Guerry Patricia
Published: October 2006

A method of insertional mutagenesis for naturally transformable organisms has been adapted from Haemophilus influenzae and applied to the study of the pathogenesis of Campylobacter jejuni. A series of kanamycin‐resistant Insertional mutants of C. jejuni 81–176 has been generated and screened for loss of ability to invade INT407 cells. Eight noninvasive mutants were identified which showed 18‐200‐fold reductions in the level of invasion compared with the parent. Three of these eight show defects in motility, and five are fully motile. The three mutants with motility defects were further characterized to evaluate the method. One mutant, K2–32, which is non‐adherent and non‐invasive, has an insertion of the kanamycin‐resistance cassette into the flaA flagellin gene and has greatly reduced motility and a truncated flagellar filament typical of flaA mutants. The adherent non‐invasive mutants K2–37 and K2–55 are phenotypically paralysed, i.e. they have a full‐length flagellar filament but are non‐motile. All three mutants show an aberration in flagellar structure at the point at which the filament attaches to the cell. Mutants K2–37 and K2–55 represent overlapping deletions affecting the same gene, termed pflA (paralysed flagella). This gene encodes a predicted protein of 788 amino acid residues and a molecular weight of 90 977 with no significant homology to known proteins. Site‐specific insertional mutants into this open reading frame result in the same paralysed flagellar phenotype and the same invasion defects as the original mutants.
Amoebapores, a family of membranolytic peptides from cytoplasmic granules of Entamoeba histolytica: isolation, primary structure, and pore bacterial cytoplasmic membranes
Authors: Leippe Matthias , Andrä Jörg , Nickel Rose , Tannich Egbert , Muller‐Eberhard Hans J.
Published: October 2006

Three peptides with pore‐forming activity were isolated from the cytoplasmic granules of pathogenic Entamoeba histolytica by acidic extraction, gel filtration and reversed‐phase high‐performance liquid chromatography. Partial amino acid sequence analysis of the three active peptides revealed that the most abundant of them was amoebapore and the other two were isoforms thereof. Cloning and sequencing of genomic DNA resolved the amino acid sequence of the two newly recognized peptides. The three peptides designated amoebapores A, B and C were found to have the same molecular size but to differ markedly in their primary structure, although all six cysteine residues are conserved. Despite sequence divergence, structural implications predict for the three peptides a similar amphipathic α‐helical conformation stabilized by disulphide bonds. All three isoforms exhibit pore‐forming activity toward lipid vesicles, but they differ in their kinetics. They also are capable of perturbing the integrity of bacterial cytoplasmic membranes and thereby kill Gram‐positive bacteria. The amoebapores represent a distinct family of membrane‐active peptides that may function intracellularly as antimicrobial agents but may also confer cytolytic activity on the parasite.