- Volume 149, Issue 11, 2003
Volume 149, Issue 11, 2003
- Review
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Type II protein secretion and its relationship to bacterial type IV pili and archaeal flagella
Homologues of the protein constituents of the Klebsiella pneumoniae (Klebsiella oxytoca) type II secreton (T2S), the Pseudomonas aeruginosa type IV pilus/fimbrium biogenesis machinery (T4P) and the Methanococcus voltae flagellum biogenesis machinery (Fla) have been identified. Known constituents of these systems include (1) a major prepilin (preflagellin), (2) several minor prepilins (preflagellins), (3) a prepilin (preflagellin) peptidase/methylase, (4) an ATPase, (5) a multispanning transmembrane (TM) protein, (6) an outer-membrane secretin (lacking in Fla) and (7) several functionally uncharacterized envelope proteins. Sequence and phylogenetic analyses led to the conclusion that, although many of the protein constituents are probably homologous, extensive sequence divergence during evolution clouds this homology so that a common ancestry can be established for all three types of systems for only two constituents, the ATPase and the TM protein. Sequence divergence of the individual T2S constituents has occurred at characteristic rates, apparently without shuffling of constituents between systems. The same is probably also true for the T4P and Fla systems. The family of ATPases is much larger than the family of TM proteins, and many ATPase homologues function in capacities unrelated to those considered here. Many phylogenetic clusters of the ATPases probably exhibit uniform function. Some of these have a corresponding TM protein homologue although others probably function without one. It is further shown that proteins that compose the different phylogenetic clusters in both the ATPase and the TM protein families exhibit unique structural characteristics that are of probable functional significance. The TM proteins are shown to have arisen by at least two dissimilar intragenic duplication events, one in the bacterial kingdom and one in the archaeal kingdom. The archaeal TM proteins are twice as large as the bacterial TM proteins, suggesting an oligomeric structure for the latter.
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- Microbiology Comment
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- Cell And Developmental Biology
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Time series analysis demonstrates the absence of pulsatile hyphal growth
More LessHyphal tip growth has been previously reported as pulsatile, defined as regularly alternating fast and slow rates of extension. The growth of pollen tubes, and hyphae of Neurospora crassa and Saprolegnia ferax were analysed using high spatial and temporal resolution. By using long (100–500 s) records of growth rate, sampled every second, it was possible to apply rigorous statistical analysis of the time series. As previously demonstrated, pollen tubes can show pulsatile growth, detectable with this system. In contrast, hyphal growth rates do not show any evidence of pulsatile growth; instead, growth rates appear to fluctuate randomly. It is concluded that pulsatile growth is not a common feature of hyphal tip growth.
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In situ localization of manganese peroxidase production in mycelial pellets of Phanerochaete chrysosporium
More LessThe ultrastructure of Phanerochaete chrysosporium hyphae from pellets in submerged liquid cultures was investigated in order to learn more about the interrelation between fungal architecture and manganese peroxidase (MnP) production. At day 2 of cultivation, some subapical regions of hyphae in the outer and middle zones of the pellet initiated differentiation into intercalary thick-walled chlamydospore-like cells of about 10 μm diameter. At the periphery of the cytoplasm of these cells, a large number of mitochondria and Golgi-like vesicles were observed. The sites of MnP production were localized at different stages of cultivation by an immunolabelling procedure. The immunomarker of MnP was mainly concentrated in the chlamydospore-like cells and principally distributed in Golgi-like vesicles located at the periphery of the cytoplasm. The apices of hyphae in the outer layer of the pellets were apparently minor sites of MnP production. Maximal MnP release into the culture supernatant coincided with apparent autolysis of the chlamydospore-like cells. Production of extracellular autolytic chitinase and protease coincided with the disappearance of these structures from the pellets. The chlamydospore-like cells observed in the mycelial pellets of P. chrysosporium could be metabolically active entities operating as an enzyme reservoir, delivering their content into the surrounding medium possibly by an enzyme-mediated autolytic process.
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- Biochemistry And Molecular Biology
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Foreign signal peptides can constitute a barrier to functional expression of periplasmic proteins in Haemophilus influenzae
More LessTo study the periplasmic branch of iron (ferric ion) uptake systems in Gram-negative bacteria, genetic reconstitution experiments were initiated in Haemophilus influenzae involving exchange of the periplasmic iron-binding protein. The expression of many of the heterologous periplasmic ferric-binding proteins (FbpAs) was quite limited. Transformation experiments with the fbpA gene from Neisseria gonorrhoeae yielded two colony sizes with different phenotypic characteristics. The small colonies contained the intact N. gonorrhoeae fbpA gene and were deficient in utilization of transferrin iron. The large colonies contained hybrid H. influenzae/N. gonorrhoeae fbpA genes, were proficient in transferrin iron utilization and had enhanced levels of expression of FbpA. These hybrid genes included several that encoded the mature N. gonorrhoeae FbpA with the H. influenzae signal peptide. To more fully evaluate the effect of foreign signal peptides, a series of hybrid genes were prepared that exchanged the signal peptides from H. influenzae FbpA, N. gonorrhoeae FbpA and the TEM-1 β-lactamase. The presence of the H. influenzae leader was required for functional expression of FbpAs and was shown to dramatically increase the level of β-lactamase activity.
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Biosynthetic specificity of the rhamnosyltransferase gene of Mycobacterium avium serovar 2 as determined by allelic exchange mutagenesis
In prior studies, through recombinant expression in Mycobacterium smegmatis, the rtfA gene of Mycobacterium avium was shown to encode a rhamnosyltransferase that catalyses the addition of rhamnose (Rha) to the 6-deoxytalose of serovar 2-specific glycopeptidolipid (GPL). Whether RtfA also catalyses the transfer of Rha to the alaninol of the lipopeptide core is unknown. An isogenic rtfA mutant of M. avium serovar 2 strain TMC724 was derived using a novel allelic exchange mutagenesis system utilizing a multicopy plasmid that contained the katG gene of Mycobacterium bovis and the gene encoding green fluorescent protein (gfp). Overexpression of KatG in M. avium resulted in increased susceptibility to isoniazid, thus providing counter-selection by enriching for clones that had lost plasmid DNA. Plasmid loss was confirmed by screening for gfp-negative clones to select putative allelic exchange mutants. Two exchange mutants were created, confirmed by Southern hybridization, and demonstrated loss of serovar 2-specific GPL by thin-layer chromatography (TLC). Gas chromatography of alditol acetate derivatives revealed the loss of Rha and the terminal 2,3-O-Me-fucose and preservation of 3-O-Me-Rha and 3,4-O-Me-Rha substituents at the terminal alaninol of the lipopeptide core. Complementation of rtfA in trans through an integrative plasmid restored serovar 2-specific GPL expression identical to wild-type TMC724. This result shows that rtfA encodes an enzyme responsible only for the transfer of Rha to the serovar 2-specific oligosaccharide and provides a system of allelic exchange for M. avium as a tool for future genetic studies involving this species.
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Ammonium utilization in Bacillus subtilis: transport and regulatory functions of NrgA and NrgB
More LessBacillus subtilis uses glutamine as the best source of nitrogen. In the absence of glutamine, alternative nitrogen sources such as ammonium can be used. Ammonium utilization involves the uptake of the gas or the ammonium ion, the synthesis of glutamine by the glutamine synthetase and the recycling of the glutamate by the glutamate synthase. In this work, ammonium transport in B. subtilis was studied. At high ammonium concentrations, a large fraction of the ammonium is present as ammonia, which may enter the cell via diffusion. In contrast, the ammonium transporter NrgA is required for ammonium utilization at low concentrations or at low pH values when the equilibrium between uncharged ammonia and the ammonium ion is shifted towards ammonium. Moreover, a functional NrgA is essential for the transport of the ammonium analogue methylammonium. NrgA is encoded in the nrgAB operon. The product of the second gene, NrgB, is a member of the PII family of regulatory proteins. In contrast to PII proteins from other organisms, there is no indication for a covalent modification of NrgB in response to the nitrogen supply of the cell. It is demonstrated here that NrgB is localized at the membrane, most likely in association with the ammonium transporter NrgA. The presence of a functional NrgB is required for full-level expression of the nrgAB operon in response to nitrogen limitation, suggesting that NrgB might relay the information on ammonium availability to downstream regulatory factors and thus fine-tune their activity.
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An aberrant protein synthesis activity is linked with antibiotic overproduction in rpsL mutants of Streptomyces coelicolor A3(2)
More LessCertain mutations in the rpsL gene (encoding the ribosomal protein S12) activate or enhance antibiotic production in various bacteria. K88E and P91S rpsL mutants of Streptomyces coelicolor A3(2), with an enhanced actinorhodin production, were found to exhibit an aberrant protein synthesis activity. While a high level of this activity (as determined by the incorporation of labelled leucine) was detected at the late stationary phase in the mutants, it decreased with age of the cells in the wild-type strain. In addition, the aberrant protein synthesis was particularly pronounced when cells were subjected to amino acid shift-down, and was independent of their ability to accumulate ppGpp. Ribosomes of K88E and P91S mutants displayed an increased accuracy in protein synthesis as demonstrated by the poly(U)-directed cell-free translation system, but so did K43N, K43T, K43R and K88R mutants, which were streptomycin resistant but showed no effect on actinorhodin production. This eliminates the possibility that the increased accuracy level is a cause of the antibiotic overproduction in the K88E and P91S mutants. The K88E and P91S mutant ribosomes exhibited an increased stability of the 70S complex under low concentrations of magnesium. The authors propose that the aberrant activation of protein synthesis caused by the increased stability of the ribosome is responsible for the remarkable enhancement of antibiotic production in the K88E and P91S mutants.
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Tetra-amino-acid tandem repeats are involved in HsdS complementation in type IC restriction–modification systems
More LessAll known type I restriction and modification (R–M) systems of Escherichia coli and Salmonella enterica belong to one of four discrete families: type IA, IB, IC or ID. The classification of type I systems from a wide range of other genera is mainly based on complementation and molecular evidence derived from the comparison of the amino acid similarity of the corresponding subunits. This affiliation was seldom based on the strictest requirement for membership of a family, which depends on relatedness as demonstrated by complementation tests. This paper presents data indicating that the type I NgoAV R–M system from Neisseria gonorrhoeae, despite the very high identity of HsdM and HsdR subunits with members of the type IC family, does not show complementation with E. coli type IC R–M systems. Sequence analysis of the HsdS subunit of several different potential type IC R–M systems shows that the presence of different tetra-amino-acid sequence repeats, e.g. TAEL, LEAT, SEAL, TSEL, is characteristic for type IC R–M systems encoded by distantly related bacteria. The other regions of the HsdS subunits potentially responsible for subunit interaction are also different between a group of distantly related bacteria, but show high similarity within these bacteria. Complementation between the NgoAV R–M system and members of the EcoR124 R–M family can be restored by changing the tetra-amino-acid repeat within the HsdS subunit. The authors propose that the type IC family of R–M systems could consist of several complementation subgroups whose specificity would depend on differences in the conserved regions of the HsdS polypeptide.
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(De)regulation of key enzyme steps in the shikimate pathway and phenylalanine-specific pathway of the actinomycete Amycolatopsis methanolica
More LessPrephenate dehydratase (PDT), chorismate mutase (CM) and 3-deoxy-d-arabino-7-heptulosonate 7-phosphate (DAHP) synthase are key regulatory enzymes in aromatic amino acid biosynthesis in the actinomycete Amycolatopsis methanolica. Deregulated, feedback-control-resistant mutants were isolated by incubation of A. methanolica on glucose mineral agar containing the toxic analogue p-fluoro-dl-phenylalanine (pFPhe). Several of these mutants had completely lost PDT sensitivity to Phe inhibition and Tyr activation. Mutant characterization yielded new information about PDT amino acid residues involved in Phe and Tyr effector binding sites. A. methanolica wild-type cells grown on glucose mineral medium normally possess a bifunctional CM/DAHP synthase protein complex (with DS1, a plant-type DAHP synthase). The CM activity of this protein complex is feedback-inhibited by Tyr and Phe, while DS1 activity is mainly inhibited by Trp. Isolation of pFPhe-resistant mutants yielded two feedback-inhibition-resistant CM mutants. These were characterized as regulatory mutants, derepressed in (a) synthesis of CM, now occurring as an abundant, feedback-inhibition-resistant, separate protein, and (b) synthesis of an alternative DAHP synthase (DS2, an E. coli-type DAHP synthase), only inhibited by Tyr and Trp. DS1 and DS2 thus are well integrated in A. methanolica primary metabolism: DS1 and CM form a protein complex, which stimulates CM activity and renders it sensitive to feedback inhibition by Phe and Tyr. Synthesis of CM and DS2 proteins appears to be controlled co-ordinately, sensitive to Phe-mediated feedback repression.
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Characterization of the LlaCI methyltransferase from Lactococcus lactis subsp. cremoris W15 provides new insights into the biology of type II restriction–modification systems
More LessThe gene encoding the LlaCI methyltransferase (M.LlaCI) from Lactococcus lactis subsp. cremoris W15 was overexpressed in Escherichia coli. The enzyme was purified to apparent homogeneity using three consecutive steps of chromatography on phosphocellulose, blue-agarose and Superose 12HR, yielding a protein of M r 31 300±1000 under denaturing conditions. The exact position of the start codon AUG was determined by protein microsequencing. This enzyme recognizes the specific palindromic sequence 5′-AAGCTT-3′. Purified M.LlaCI was characterized. Unlike many other methyltransferases, M.LlaCI exists in solution predominantly as a dimer. It modifies the first adenine residue at the 5′ end of the specific sequence to N 6-methyladenine and thus is functionally identical to the corresponding methyltransferases of the HindIII (Haemophilus influenzae Rd) and EcoVIII (Escherichia coli E1585-68) restriction–modification systems. This is reflected in the identity of M.LlaCI with M.HindIII and M.EcoVIII noted at the amino acid sequence level (50 % and 62 %, respectively) and in the presence of nine sequence motifs conserved among N 6-adenine β-class methyltransferases. However, polyclonal antibodies raised against M.EcoVIII cross-reacted with M.LlaCI but not with M.HindIII. Restriction endonucleases require Mg2+ for phosphodiester bond cleavage. Mg2+ was shown to be a strong inhibitor of the M.LlaCI enzyme and its isospecific homologues. This observation suggests that sensitivity of the M.LlaCI to Mg2+ may strengthen the restriction activity of the cognate endonuclease in the bacterial cell. Other biological implications of this finding are also discussed.
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- Environmental Microbiology
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3- and 4-alkylphenol degradation pathway in Pseudomonas sp. strain KL28: genetic organization of the lap gene cluster and substrate specificities of phenol hydroxylase and catechol 2,3-dioxygenase
More LessThe enzymes and genes responsible for the catabolism of higher alkylphenols have not been characterized in aerobic bacteria. Pseudomonas sp. strain KL28 can utilize a wide range of alkylphenols, which include the 4-n-alkylphenols (C1–C5). The genes, designated as lap (for long-chain alkylphenols), encoding enzymes for the catabolic pathway were cloned from chromosomal DNA and sequenced. The lap genes are located in a 13·2 kb region with 14 ORFs in the order lapRBKLMNOPCEHIFG and with the same transcriptional orientation. The lapR gene is transcribed independently and encodes a member of the XylR/DmpR positive transcriptional regulators. lapB, the first gene in the lap operon, encodes catechol 2,3-dioxygenase (C23O). The lapKLMNOP and lapCEHIFG genes encode a multicomponent phenol hydroxylase (mPH) and enzymes that degrade derivatives of 2-hydroxymuconic semialdehyde (HMS) to TCA cycle intermediates, respectively. The PlapB promoter contains motifs at positions −24(GG) and −12(GC) which are typically found in σ 54-dependent promoters. A promoter assay using a PlapB : : gfp transcriptional fusion plasmid showed that lapB promoter activity is inducible and that it responds to a wide range of (alkyl)phenols. The structural genes encoding enzymes required for this catabolism are similar (42–69 %) to those encoded on a catabolic pVI150 plasmid from an archetypal phenol degrader, Pseudomonas sp. CF600. However, the lap locus does not include genes encoding HMS hydrolase and ferredoxin. The latter is known to be functionally associated with C23O for use of 4-alkylcatechols as substrates. The arrangement of the lap catabolic genes is not commonly found in other meta-cleavage operons. Substrate specificity studies show that mPH preferentially oxidizes 3- and 4-alkylphenols to 4-alkylcatechols. C23O preferentially oxidizes 4-alkylcatechols via proximal (2,3) cleavage. This indicates that these two key enzymes have unique substrate preferences and lead to the establishment of the initial steps of the lap pathway in strain KL28.
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Characterization of a soil-derived bacterial consortium degrading 4-chloroaniline
More LessA bacterial consortium comprising four different species was isolated from an Indonesian agricultural soil using a mixture of aniline and 4-chloroaniline (4CA) as principal carbon sources. The four species were identified as Chryseobacterium indologenes SB1, Comamonas testosteroni SB2, Pseudomonas corrugata SB4 and Stenotrophomonas maltophilia SB5. Growth studies on aniline and 4CA as single and mixed substrates demonstrated that the bacteria preferred to grow on and utilize aniline rather than 4CA, although both compounds were eventually depleted from the culture supernatant. However, despite 100 % disappearance of the parent substrates, the degradation of 4CA was always characterized by incomplete dechlorination and 4-chlorocatechol accumulation. This result suggests that further degradation of 4-chlorocatechol may be the rate-limiting step in the metabolism of 4CA by the bacterial consortium. HPLC-UV analysis showed that 4-chlorocatechol was further degraded via an ortho-cleavage pathway by the bacterial consortium. This hypothesis was supported by the results from enzyme assays of the crude cell extract of the consortium revealing catechol 1,2-dioxygenase activity which converted catechol and 4-chlorocatechol to cis,cis-muconic acid and 3-chloro-cis,cis-muconic acid respectively. However, the enzyme had a much higher conversion rate for catechol [156 U (g protein)−1] than for 4-chlorocatechol [17·2 U (g protein)−1], indicating preference for non-chlorinated substrates. Members of the bacterial consortium were also characterized individually. All isolates were able to assimilate aniline. P. corrugata SB4 was able to grow on 4CA solely, while S. maltophilia SB5 was able to grow on 4-chlorocatechol. These results suggest that the degradation of 4CA in the presence of aniline by the bacterial consortium was a result of interspecies interactions.
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- Genes And Genomes
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Transcriptional regulation of Pseudomonas aeruginosa rhlR, encoding a quorum-sensing regulatory protein
More LessThe Pseudomonas aeruginosa rhlR gene encodes the transcriptional regulator RhlR which has a central role in the quorum-sensing response. Different gene products involved in bacterial pathogenesis are regulated at the transcriptional level by two quorum-sensing response systems, Las and Rhl. The expression of rhlR has been reported to be under the control of the Las system, but its transcriptional regulation has not been studied in detail. Here, the rhlR promoter region has been characterized and shown to present four different transcription start sites, two of which are included in the upstream gene (rhlB) coding region. It was found that rhlR expression is not only dependent on LasR but also on different regulatory proteins such as Vfr and RhlR itself, and also on the alternative sigma factor σ 54. It is reported that rhlR expression is partially LasR-independent under certain culture conditions and is strongly influenced by environmental factors.
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A novel type of DNA curvature present in a Clostridium perfringens ferredoxin gene: characterization and role in gene expression
This study has revealed that a Clostridium perfringens ferredoxin gene (per-fdx) possesses a novel type of DNA curvature, which is formed by five phased A-tracts extending from upstream to downstream of the −35 region. The three A-tracts upstream of the promoter and the two within the promoter are located at the positions corresponding to A-tracts present in a C. perfringens phospholipase C gene (plc) and a Clostridium pasteurianum ferredoxin gene (pas-fdx), respectively. DNA fragments of the per-fdx, pas-fdx and plc genes (nucleotide positions −69 to +1 relative to the transcription initiation site) were fused to a chloramphenicol acetyltransferase reporter gene on a plasmid, pPSV, and their in vivo promoter activities were examined by assaying the chloramphenicol acetyltransferase activity of each C. perfringens transformant. Comparison of the three constructs showed that the order of promoter activity is, in descending order, per-fdx, pas-fdx and plc. Deletion of the three upstream A-tracts of the per-fdx gene drastically decreased the promoter activity, as demonstrated previously for the plc promoter. Substitution of the most downstream A-tract decreased the promoter activities of the per-fdx and pas-fdx genes. These results indicate that not only the phased A-tracts upstream of the promoter but also those within the promoter stimulate the promoter activity, and suggest that the high activity of the per-fdx promoter is due to the combined effects of these two types of A-tracts.
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Intron requirement for AFP gene expression in Trichoderma viride
Jun Xu and Zhen Zhen GongThe 430 bp ORF of the Aspergillus giganteus antifungal protein (AFP) gene, containing two small introns, was fused between the promoter and the terminator of the Aspergillus nidulans trpC gene. The AFP gene in this vector produced detectable levels of spliced mRNA in Trichoderma viride. In contrast, in the same vector configuration, its 285 bp intronless derivative showed no accumulation of mRNA when transformed into T. viride. Such expression results were confirmed at the protein level. This fact demonstrated that the introns were required for AFP gene expression in T. viride. This is thought to be a novel phenomenon found in filamentous fungi. Although the mechanism of splicing in filamentous fungi might be similar to that in other eukaryotes, little is known of how it affects expression. This study suggests that the small introns in filamentous fungal genes may not only act as intervening elements, but may also play crucial roles in gene expression by affecting mRNA accumulation. Furthermore, it may provide new evidence for intron-dependent evolution.
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Cryptons: a group of tyrosine-recombinase-encoding DNA transposons from pathogenic fungi
More LessA new group of transposable elements, which the authors have named cryptons, was detected in several pathogenic fungi, including the basidiomycete Cryptococcus neoformans, and the ascomycetes Coccidioides posadasii and Histoplasma capsulatum. These elements are unlike any previously described transposons. An archetypal member of the group, crypton Cn1, is 4 kb in length and is present at a low but variable copy number in a variety of C. neoformans strains. It displays interstrain variations in its insertion sites, suggesting recent mobility. The internal region contains a long gene, interrupted by several introns. The product of this gene contains a putative tyrosine recombinase near its middle, and a region similar in sequence to the DNA-binding domains of several fungal transcription factors near its C-terminus. The element contains no long repeat sequences, but is bordered by short direct repeats which may have been produced by its insertion into the host genome by recombination. Many of the structural features of crypton Cn1 are conserved in the other known cryptons, suggesting that these elements represent the functional forms. The presence of cryptons in ascomycetes and basidiomycetes suggests that this is an ancient group of elements (>400 million years old). Sequence comparisons suggest that cryptons may be related to the DIRS1 and Ngaro1 groups of tyrosine-recombinase-encoding retrotransposons.
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The 64 508 bp IncP-1β antibiotic multiresistance plasmid pB10 isolated from a waste-water treatment plant provides evidence for recombination between members of different branches of the IncP-1β group
More LessThe complete 64 508 bp nucleotide sequence of the IncP-1β antibiotic-resistance plasmid pB10, which was isolated from a waste-water treatment plant in Germany and mediates resistance against the antimicrobial agents amoxicillin, streptomycin, sulfonamides and tetracycline and against mercury ions, was determined and analysed. A typical class 1 integron with completely conserved 5′ and 3′ segments is inserted between the tra and trb regions. The two mobile gene cassettes of this integron encode a β-lactamase of the oxacillin-hydrolysing type (Oxa-2) and a gene product of unknown function (OrfE-like), respectively. The pB10-specific gene load present between the replication module (trfA1) and the origin of vegetative replication (oriV) is composed of four class II (Tn3 family) transposable elements: (i) a Tn501-like mercury-resistance (mer) transposon downstream of the trfA1 gene, (ii) a truncated derivative of the widespread streptomycin-resistance transposon Tn5393c, (iii) the insertion sequence element IS1071 and (iv) a Tn1721-like transposon that contains the tetracycline-resistance genes tetA and tetR. A very similar Tn501-like mer transposon is present in the same target site of the IncP-1β degradative plasmid pJP4 and the IncP-1β resistance plasmid R906, suggesting that pB10, R906 and pJP4 are derivatives of a common ancestor. Interestingly, large parts of the predicted pB10 restriction map, except for the tetracycline-resistance determinant, are identical to that of R906. It thus appears that plasmid pB10 acquired as many as five resistance genes via three transposons and one integron, which it may rapidly spread among bacterial populations given its high promiscuity. Comparison of the pB10 backbone DNA sequences with those of other sequenced IncP-1β plasmids reveals a mosaic structure. While the conjugative transfer modules (trb and tra regions) and the replication module are very closely related to the corresponding segments of the IncP-1β resistance plasmid R751 and even more similar to the IncP-1β degradative plasmids pTSA and pADP-1, the stable inheritance operons klcAB–korC and kleAEF are most similar to those of the IncP-1β resistance plasmid pB4, and clearly less similar to the other IncP-1β plasmids. This suggests that IncP-1β plasmids can undergo recombination in the environment, which may enhance plasmid diversity and bacterial adaptability.
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- Pathogens And Pathogenicity
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The role of lex2 in lipopolysaccharide biosynthesis in Haemophilus influenzae strains RM7004 and RM153
The locus lex2, comprising lex2A and lex2B, contributes to the phase-variable expression of lipopolysaccharide (LPS) of Haemophilus influenzae and was found to be present in 74 % of strains investigated. lex2A contains 5′-GCAA repeats which vary in number from 4 to 46 copies between strains. The locus was cloned from the serotype b strains RM7004 and RM153 and showed >99 % nucleotide sequence identity between these strains and the published lex2 sequence. Disruption of the lex2B gene in strain RM7004 resulted in truncation of some LPS glycoforms, shown by gel fractionation, with only one glycoform reacting with a digalactoside-specific monoclonal antibody, 4C4, compared with four LPS glycoforms in the more elongated LPS of the parent strain. Mass spectrometry and NMR analyses of LPS from the lex2B mutant revealed loss of the terminal digalactoside as well as the second β-glucose extending from the first heptose of the inner core. The authors conclude that Lex2B is the β-(1-4)-glucosyltransferase that adds the second β-glucose to the first β-glucose as part of the oligosaccharide extension from the first heptose of the LPS of strain RM7004. Investigation of the expression of the lex2 locus indicated that the genes are co-transcribed and that both reading frames are required for addition of this second β-glucose in a phase-variable manner.
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Genetic analysis of a plasmid encoding haemocin production in Haemophilus paragallinarum
More LessThe full sequence of plasmid p250, isolated from Haemophilus paragallinarum strain HP250, has been obtained. The plasmid contains seven ORFs: a putative integrase, a putative replication protein (repB) and five ORFs similar to those from the haemocin (bacteriocin) hmcDCBAI operon from Haemophilus influenzae. Of 19 other non-plasmid-containing H. paragallinarum strains screened (11 serovar reference strains and 8 field isolates), 17 strains produced haemocin and were resistant to killing by strain HP250. These strains, unlike strain HP250, have a chromosomally encoded haemocin operon. A number of other members of the family Pasteurellaceae were tested for haemocin sensitivity. Pasteurella avium, Pasteurella volantium and Pasteurella species A, all non-pathogenic bacteria found in the respiratory tract of chickens suffering from respiratory diseases, were sensitive to H. paragallinarum haemocin. However, amongst the pathogenic Pasteurellaceae, 50 % of P. multocida isolates and all five isolates of Pasteurella haemolytica tested were sensitive to the haemocin. Given the prevalence of haemocin production in H. paragallinarum strains, it may play a role in aiding colonization by inhibiting other Gram-negative bacteria that are associated with the respiratory tract in chickens. The origin of replication from plasmid p250 has been used to generate an Escherichia coli–H. paragallinarum shuttle vector which may be useful in genetically manipulating H. paragallinarum.
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The aquaporin gene aqpX of Brucella abortus is induced in hyperosmotic conditions
More LessAn aquaporin gene (aqpX) was previously detected in the pathogenic bacterium Brucella abortus. Earlier studies showed that AqpX mediated rapid and large water fluxes in both directions in response to sudden osmotic up- or downshifts. Here, to study the role and the expression of the aqpX gene in B. abortus, an aqpX null mutant was constructed using an aqpX : : lacZ gene fusion. This mutant showed no significant difference in growth rate compared to the wild-type strain when grown in rich and minimal media, demonstrating that disruption of the aqpX gene was not lethal for B. abortus. The role of the B. abortus AqpX water channel was investigated by exposing the cells to hypo- and hyperosmolar conditions. While in hyperosmolar environments the growth rate of the knockout mutant was not affected, in hypo-osmolar conditions this mutant showed reduced viability after 50 h of growth. β-Galactosidase assays and RT-PCR revealed that aqpX gene expression and the amount of aqpX mRNA were markedly increased in hyperosmolar conditions. Moreover, B. abortus aqpX expression levels were enhanced during the mid-exponential phase of growth. These results indicated that the expression of aqpX was regulated during the growth curve and induced in hyperosmolar conditions. This report is believed to be the first example of the induction of a bacterial aquaporin in hypertonic conditions.
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Different susceptibility of two animal species infected with isogenic mutants of Mycobacterium bovis identifies phoT as having roles in tuberculosis virulence and phosphate transport
More LessThe Mycobacterium tuberculosis complex includes Mycobacterium bovis, which causes tuberculosis in most mammals, including humans. In previous work, it was shown that M. bovis ATCC 35721 has a mutation in its principal sigma factor gene, sigA, causing a single amino acid change affecting binding of SigA with the accessory transcription factor WhiB3. ATCC 35721 is avirulent when inoculated subcutaneously into guinea pigs but can be restored to virulence by integration of wild-type sigA to produce M. bovis WAg320. Subsequently, it was surprising to discover that WAg320 was not virulent when inoculated intratracheally into the Australian brushtail possum (Trichosurus vulpecula), a marsupial that is normally very susceptible to infection with M. bovis. In this study, an in vivo complementation approach was used with ATCC 35721 to produce M. bovis WAg322, which was virulent in possums, and to identify the virulence-restoring gene, phoT. There are two point deletions in the phoT gene of ATCC 35721 causing frameshift inactivation, one of which is also in the phoT of BCG. Knockout of phoT from ATCC 35723, a virulent strain of M. bovis, produced M. bovis WAg758, which was avirulent in both guinea pigs and possums, confirming that phoT is a virulence gene. The effect on virulence of mode of infection versus animal species susceptibility was investigated by inoculating all the above strains by aerosol into guinea pigs and mice and comparing these to the earlier results. Characterization of PhoT indicated that it plays a role in phosphate uptake at low phosphate concentrations. At least in vitro, this role requires the presence of a wild-type sigA gene and appears separate from the ability of phoT to restore virulence to ATCC 35721. This study shows the advantages of using different animal models as tools for the molecular biological investigation of tuberculosis virulence.
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Koch's Bacillus – a look at the first isolate of Mycobacterium tuberculosis from a modern perspective
More LessUsing molecular methods the authors have studied mycobacterial DNA taken from a 19th century victim of tuberculosis. This was the case from which Robert Koch first isolated and cultured the organism responsible for tuberculosis. The mycobacteria were preserved within five glass culture tubes as abundant bacterial colonies on slopes of a gelatinous culture medium of unknown composition. Originally presented by Koch to surgical laryngologist Walter Jobson Horne in London in 1901, the relic has, since 1983, been in the care of the Royal College of Surgeons of England. Light and electron microscopy established the presence of acid-fast mycobacteria but showed that morphological preservation was generally poor. Eleven different genomic loci were successfully amplified by PCR. This series of experiments confirmed that the organisms were indeed Mycobacterium tuberculosis and further showed that the original strain was in evolutionary terms similar to ‘modern’ isolates, having undergone the TB D1 deletion. Attempts to determine the genotypic group of the isolate were only partially successful, due in part to the degraded nature of the DNA and possibly also to a truncation in the katG gene, which formed part of the classification scheme. Spoligotyping resulted in amplification of DR spacers consistent with M. tuberculosis but with discrepancies between independent extracts, stressing the limitations of this typing method when applied to poorly preserved material.
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A Caenorhabditis elegans model of Yersinia infection: biofilm formation on a biotic surface
More LessTo investigate Yersinia pathogenicity and the evolutionary divergence of the genus, the effect of pathogenic yersiniae on the model organism Caenorhabditis elegans was studied. Three strains of Yersinia pestis, including a strain lacking pMT1, caused blockage and death of C. elegans; one strain, lacking the haemin storage (hms) locus, caused no effect. Similarly, 15 strains of Yersinia enterocolitica caused no effect. Strains of Yersinia pseudotuberculosis showed different levels of pathogenicity. The majority of strains (76 %) caused no discernible effect; 5 % caused a weak infection, 9·5 % an intermediate infection, and 9·5 % a severe infection. There was no consistent relationship between serotype and severity of infection; nor was there any relationship between strains causing infection of C. elegans and those able to form a biofilm on an abiotic surface. Electron microscope and cytochemical examination of infected worms indicated that the infection phenotype is a result of biofilm formation on the head of the worm. Seven transposon mutants of Y. pseudotuberculosis strain YPIII pIB1 were completely or partially attenuated; mutated genes included genes encoding proteins involved in haemin storage and lipopolysaccharide biosynthesis. A screen of 15 defined C. elegans mutants identified four where mutation caused (complete) resistance to infection by Y. pseudotuberculosis YPIII pIB1. These mutants, srf-2, srf-3, srf-5 and the dauer pathway gene daf-1, also exhibit altered binding of lectins to the nematode surface. This suggests that biofilm formation on a biotic surface is an interactive process involving both bacterial and invertebrate control mechanisms.
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Proteus mirabilis fimbriae (PMF) are important for both bladder and kidney colonization in mice
Proteus mirabilis expresses different types of fimbriae simultaneously. Several fimbrial types have been described and their role in the colonization of the urinary tract is under study. Previously, P. mirabilis fimbriae (PMF) have been shown to be associated with bacterial colonization of the lower urinary tract but not of the kidneys. In this study, a pmfA mutant was generated and used in several in vivo and in vitro studies. Two different urinary tract infection models in the mouse and two in vitro assays of bacterial adhesion to uroepithelial cells were performed. Expression of PmfA in a collection of P. mirabilis strains of different sources was also assessed. The results shown here indicate that PMF are involved in both bladder and kidney colonization by P. mirabilis and that these fimbriae are widely distributed among P. mirabilis isolates from different origins since all strains tested expressed PmfA.
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Inhibition of adhesion of yeasts and bacteria by poly(ethylene oxide)-brushes on glass in a parallel plate flow chamber
More LessPoly(ethylene oxide) (PEO)-brushes are generally recognized as protein-repellent surfaces, and although a role in discouraging microbial adhesion has been established for some strains and species, no study exists on the effects of PEO-brushes on a large variety of bacterial and yeast strains. In this paper, a PEO-brush has been covalently attached to glass and silica by reaction in a polymer melt. Subsequently, the presence of a PEO-brush was demonstrated using contact angle measurements, X-ray photoelectron spectroscopy and ellipsometry. For five bacterial (Staphylococcus epidermidis, Staphylococcus aureus, Streptococcus salivarius, Escherichia coli and Pseudomonas aeruginosa) and two yeast strains (Candida albicans and Candida tropicalis), adhesion to PEO-brushes was compared with adhesion to bare glass in a parallel plate flow chamber. The initial deposition rates of Sta. epidermidis, Sta. aureus and Str. salivarius to glass were relatively high, between 2400 and 2600 cm−2 s−1, while E. coli and P. aeruginosa deposited much more slowly. The initial deposition rates of the yeasts to glass were 144 and 444 cm−2 s−1 for C. albicans GB 1/2 and C. tropicalis GB 9/9, respectively. Coating of the glass surface with a PEO-brush yielded more than 98 % reduction in bacterial adhesion, although for the more hydrophobic P. aeruginosa a smaller reduction was observed. For both yeast species adhesion suppression was less effective than for the bacteria and here too the more hydrophobic C. tropicalis showed less reduction than the more hydrophilic C. albicans. The PEO-brush had a thickness of 22 nm in water, as inferred from ellipsometry. Assuming that on bare glass the adhered micro-organisms are positioned only a few nanometers away from the surface and that the brush keeps them at a distance of 22 nm, it is calculated that the brush yields a sevenfold attenuation of the Lifshitz–Van der Waals attraction to the surface between the micro-organisms and the surface. Decreased Lifshitz–van der Waals attraction may be responsible for the suppression of the microbial adhesion observed.
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σ B-dependent expression patterns of compatible solute transporter genes opuCA and lmo1421 and the conjugated bile salt hydrolase gene bsh in Listeria monocytogenes
More LessListeria monocytogenes is a food-borne pathogen that can persist and grow under a wide variety of environmental conditions including low pH and high osmolarity. The alternative sigma factor σ B contributes to L. monocytogenes survival under extreme conditions. The purpose of this study was to identify and confirm specific σ B-dependent genes in L. monocytogenes and to characterize their expression patterns under various stress conditions. opuCA, lmo1421 and bsh were identified as putative σ B-dependent genes based on the presence of a predicted σ B-dependent promoter sequence upstream of each gene. opuCA and lmo1421 encode known and putative compatible solute transporter proteins, respectively, and bsh encodes a conjugated bile salt hydrolase (BSH). Reporter fusions and semi-quantitative RT-PCR techniques were used to confirm σ B-dependent regulation of these stress-response genes and to determine their expression patterns in response to environmental stresses. RT-PCR demonstrated that opuCA, lmo1421 and bsh transcript levels are reduced in stationary-phase L. monocytogenes ΔsigB cells relative to levels present in wild-type cells. Furthermore, BSH activity is abolished in a L. monocytogenes ΔsigB strain. RT-PCR confirmed growth-phase-dependent expression of opuCA, with highest levels of expression in stationary-phase cells. The L. monocytogenes wild-type strain exhibited two- and threefold induction of opuCA expression and seven- and fivefold induction of lmo1421 expression following 10 and 15 min exposure to 0·5 M KCl, respectively, as determined by RT-PCR, suggesting rapid induction of σ B activity in exponential-phase L. monocytogenes upon exposure to salt stress. Single-copy chromosomal opuCA–gus reporter fusions also showed significant induction of opuCA expression following exposure of exponential-phase cells to increased salt concentrations (0·5 M NaCl or 0·5 M KCl). In conjunction with recent findings that indicate a role for opuCA and bsh in L. monocytogenes virulence, the data presented here provide further evidence of specific σ B-mediated contributions to both environmental stress resistance and intra-host survival in L. monocytogenes.
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- Physiology
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Chitin scar breaks in aged Saccharomyces cerevisiae
More LessAgeing in budding yeast is not determined by chronological lifespan, but by the number of times an individual cell is capable of dividing, termed its replicative capacity. As cells age they are subject to characteristic cell surface changes. Saccharomyces cerevisiae reproduces asexually by budding and as a consequence of this process both mother and daughter cell retain chitinous scar tissue at the point of cytokinesis. Daughter cells exhibit a frail structure known as the birth scar, while mother cells display a more persistent bud scar. The number of bud scars present on the cell surface is directly related to the number of times a cell has divided and thus constitutes a biomarker for replicative cell age. It has been proposed that the birth scar may be subject to stretching caused by expansion of the daughter cell; however, no previous analysis of the effect of cell age on birth or bud scar size has been reported. This paper provides evidence that scar tissue expands with the cell during growth. It is postulated that symmetrically arranged breaks in the bud scar allow these rigid chitinous structures to expand without compromising cellular integrity.
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An increase in the level of 2-oxoglutarate promotes heterocyst development in the cyanobacterium Anabaena sp. strain PCC 7120
More LessIn the filamentous cyanobacterium Anabaena sp. strain PCC 7120, a starvation of combined nitrogen induces differentiation of heterocysts, cells specialized in nitrogen fixation. How do filaments perceive the limitation of the source of combined nitrogen, and what determines the proportion of heterocysts? In cyanobacteria, 2-oxoglutarate provides a carbon skeleton for the incorporation of inorganic nitrogen. Recently, it has been proposed that the concentration of 2-oxoglutarate reflects the nitrogen status in cyanobacteria. To investigate the effect of 2-oxoglutarate on heterocyst development, a heterologous gene encoding a 2-oxoglutarate permease under the control of a regulated promoter was expressed in Anabaena sp. PCC 7120. The increase of 2-oxoglutarate within cells can trigger heterocyst differentiation in a subpopulation of filaments even in the presence of nitrate. In the absence of a source of combined nitrogen, it can increase heterocyst frequency, advance the timing of commitment to heterocyst development and further increase the proportion of heterocysts in a patS mutant. Here, it is proposed that the intracellular concentration of 2-oxoglutarate is involved in the determination of the proportion of the two cell types according to the carbon/nitrogen status of the filament.
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