RESUMO
OBJECTIVE: To perform a comprehensive review of the literature from July 2015 to June 2019 on the pathogenesis of otitis media. Bacteria, viruses and the role of the microbiome as well as the host response are discussed. Directions for future research are also suggested. DATA SOURCES: PubMed database of the National Library of Medicine. REVIEW METHODS: PubMed was searched for any papers pertaining to OM pathogenesis between July 2015 and June 2019. If in English, abstracts were assessed individually for their relevance and included in the report. Members of the panel drafted the report based on these searches and on new data presented at the 20th International Symposium on Recent Advances in Otitis Media. CONCLUSIONS: The main themes that arose in OM pathogenesis were around the need for symptomatic viral infections to develop disease. Different populations potentially having different mechanisms of pathogenesis. Novel bacterial otopathogens are emerging and need to be monitored. Animal models need to continue to be developed and used to understand disease pathogenesis. IMPLICATIONS FOR PRACTICE: The findings in the pathogenesis panel have several implications for both research and clinical practice. The most urgent areas appear to be to continue monitoring the emergence of novel otopathogens, and the need to develop prevention and preventative therapies that do not rely on antibiotics and protect against the development of the initial OM episode.
Assuntos
Infecções Bacterianas/complicações , Infecções Bacterianas/microbiologia , Microbiota , Otite Média/microbiologia , Viroses/complicações , Animais , Pesquisa Biomédica , Modelos Animais de Doenças , Orelha Média/microbiologia , Humanos , Otite Média/prevenção & controle , Otite Média/virologiaRESUMO
Nontypeable Haemophilus influenzae is a commensal that frequently causes otitis media and respiratory tract infections. The lex2 locus encodes a glycosyltransferase that is phase variably expressed and contributes to the significant intrastrain heterogeneity of lipopolysaccharide (LPS) composition in H. influenzae. In serotype b strains, Lex2B adds the second beta-glucose in the oligosaccharide extension from the proximal heptose of the triheptose inner core backbone; this extension includes a digalactoside that plays a role in resistance of the bacteria to the killing effect of serum. As part of our studies of the structure and genetics of LPS in nontypeable H. influenzae, we show here that there are allelic polymorphisms in the lex2B sequence that correlate with addition of either a glucose or a galactose to the same position in the LPS molecule across strains. Through exchange of lex2 alleles between strains we show that alteration of a single amino acid at position 157 in Lex2B appears to be sufficient to direct the alternative glucosyl- or galactosyltransferase activities. Allelic exchange strains express LPS with altered structure and biological properties compared to the wild-type LPS. Thus, Lex2B contributes to both inter- and intrastrain LPS heterogeneity through its polymorphic sequences and phase-variable expression.
Assuntos
Proteínas de Bactérias/metabolismo , Galactose/metabolismo , Glucose/metabolismo , Glicosiltransferases/metabolismo , Haemophilus influenzae/enzimologia , Lipopolissacarídeos/metabolismo , Polimorfismo Genético , Sequência de Aminoácidos , Substituição de Aminoácidos/genética , Proteínas de Bactérias/genética , Criança , DNA Bacteriano/química , DNA Bacteriano/genética , Glicosiltransferases/genética , Haemophilus influenzae/genética , Haemophilus influenzae/metabolismo , Humanos , Dados de Sequência Molecular , Alinhamento de Sequência , Análise de Sequência de DNARESUMO
Neisseria meningitidis is a major cause of bacterial septicemia and meningitis. Sequence variation of surface-exposed proteins and cross-reactivity of the serogroup B capsular polysaccharide with human tissues have hampered efforts to develop a successful vaccine. To overcome these obstacles, the entire genome sequence of a virulent serogroup B strain (MC58) was used to identify vaccine candidates. A total of 350 candidate antigens were expressed in Escherichia coli, purified, and used to immunize mice. The sera allowed the identification of proteins that are surface exposed, that are conserved in sequence across a range of strains, and that induce a bactericidal antibody response, a property known to correlate with vaccine efficacy in humans.
Assuntos
Antígenos de Bactérias/imunologia , Proteínas de Bactérias/imunologia , Vacinas Bacterianas , Genoma Bacteriano , Neisseria meningitidis/genética , Neisseria meningitidis/imunologia , Sequência de Aminoácidos , Animais , Anticorpos Antibacterianos/biossíntese , Anticorpos Antibacterianos/sangue , Antígenos de Bactérias/química , Antígenos de Bactérias/genética , Antígenos de Superfície/química , Antígenos de Superfície/genética , Antígenos de Superfície/imunologia , Cápsulas Bacterianas , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Vacinas Bacterianas/genética , Vacinas Bacterianas/imunologia , Sequência Conservada , Escherichia coli/genética , Humanos , Soros Imunes/imunologia , Camundongos , Neisseria meningitidis/classificação , Neisseria meningitidis/patogenicidade , Fases de Leitura Aberta , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/imunologia , Proteínas Recombinantes de Fusão/isolamento & purificação , Recombinação Genética , Análise de Sequência de DNA , Sorotipagem , Vacinação , VirulênciaRESUMO
The 2,272,351-base pair genome of Neisseria meningitidis strain MC58 (serogroup B), a causative agent of meningitis and septicemia, contains 2158 predicted coding regions, 1158 (53.7%) of which were assigned a biological role. Three major islands of horizontal DNA transfer were identified; two of these contain genes encoding proteins involved in pathogenicity, and the third island contains coding sequences only for hypothetical proteins. Insights into the commensal and virulence behavior of N. meningitidis can be gleaned from the genome, in which sequences for structural proteins of the pilus are clustered and several coding regions unique to serogroup B capsular polysaccharide synthesis can be identified. Finally, N. meningitidis contains more genes that undergo phase variation than any pathogen studied to date, a mechanism that controls their expression and contributes to the evasion of the host immune system.
Assuntos
Genoma Bacteriano , Neisseria meningitidis/genética , Neisseria meningitidis/patogenicidade , Análise de Sequência de DNA , Variação Antigênica , Antígenos de Bactérias/imunologia , Bacteriemia/microbiologia , Cápsulas Bacterianas/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/fisiologia , Elementos de DNA Transponíveis , Evolução Molecular , Fímbrias Bacterianas/genética , Humanos , Meningite Meningocócica/microbiologia , Infecções Meningocócicas/microbiologia , Dados de Sequência Molecular , Mutação , Neisseria meningitidis/classificação , Neisseria meningitidis/fisiologia , Fases de Leitura Aberta , Óperon , Filogenia , Recombinação Genética , Sorotipagem , Transformação Bacteriana , Virulência/genéticaRESUMO
DNA transfer between related bacterial species is enhanced by species-specific uptake sequences. These sequences have been used to identify genes that have been transferred from Haemophilus to Neisseria, providing a clear example of interspecific transfer of DNA in the evolution of the pathogenic Neisseria.
Assuntos
Evolução Molecular , Transformação Bacteriana , Bactérias/genética , Genes BacterianosRESUMO
The publication of the Haemophilus influenzae genome sequence in 1995 was a landmark in microbiological research. It has changed our understanding of the prokaryotic world, and will influence the approach and focus of research on microorganisms over the next few years. In this article we outline what has been learned from this and other genome sequencing projects, and discuss some of the potential avenues of investigation that will follow in the 'post-genome era'.
Assuntos
Genoma Bacteriano , Haemophilus influenzae/genética , Microbiologia/tendências , Evolução Biológica , Sequência de Carboidratos , Previsões , Regulação Bacteriana da Expressão Gênica , Genes Bacterianos , Técnicas Genéticas , Genética Populacional , Haemophilus influenzae/patogenicidade , Dados de Sequência Molecular , Análise de Sequência de DNARESUMO
The year 1997 saw the publication of the complete nucleotide sequence of Helicobacter pylori and Escherichia coli. It is conceivable that the complete nucleotide sequence for all the major human bacterial pathogens will be available by the end of the century. Database alignments have been used to ascribe the putative functions of open reading frames in the sequenced isolates and to define the differences between bacterial species at the nucleotide level. The most striking finding from all genome projects has been the high proportion of open reading frames that have no known function. Experimental data demonstrating the utility of the genome sequencing projects are only just beginning to emerge.
Assuntos
Genoma Bacteriano , Bactérias Gram-Negativas/genética , Bactérias Gram-Negativas/patogenicidade , Infecções por Bactérias Gram-Negativas/microbiologia , Análise de Sequência de DNA/métodos , Variação Antigênica , Bactérias Gram-Negativas/imunologia , Humanos , Virulência/genéticaRESUMO
We studied the control of proline metabolism and tryptophan biosynthesis in Streptomyces coelicolor A3(2), because proline is involved in secondary metabolism [undecylprodigiosin (Red) biosynthesis] whilst tryptophan, to our knowledge, is not. Proline transport was constitutive in wild-type cells, as were the enzymes of proline catabolism. When we analysed proline biosynthesis, we discovered that growth in the presence of proline stimulated rather than repressed the biosynthetic genes. We isolated proline transport mutants and to our surprise discovered that such strains overproduced Red. As well as losing the ability to transport proline, they had lost, to differing extents, the ability to degrade proline. However, proline biosynthesis appeared to be unaffected. It appears that proline anabolism and catabolism in S. coelicolor A3(2) is in a state of dynamic equilibrium and that if this balance is disturbed, Red biosynthesis can act as a sink for excess proline. We cloned the trpD and the trpCBA clusters of S. coelicolor A3(2) and identified a promoter within the latter cluster. This promoter appeared not to be regulated by the presence or absence of exogenous tryptophan, but rather by the growth phase and/or the growth rate of the culture. It appears, therefore, that an amino acid biosynthetic pathway that is apparently not involved in secondary metabolism in the streptomycete is regulated at the genetic level--not by feedback repression, but rather by the overall physiological state of the cell.
Assuntos
Prolina/biossíntese , Streptomyces/genética , Triptofano/biossíntese , Prolina/metabolismo , Streptomyces/metabolismo , Triptofano/metabolismoRESUMO
Mutagenesis with the transposon Tn916 was used as a strategy to identify genes required for synthesis of the Gal alpha (1-4) beta Gal component of Haemophilus influenzae strain RM7004 lipopolysaccharide. Insertion of Tn916 into an open reading frame (ORF) encoding a protein with 75% homology to the Escherichia coli methionine related protein (Mrp) is described. Mutations in mrp resulted in loss of reactivity with monoclonal antibody (mAb) 4C4, which recognises Gal alpha (1-4) beta Gal, and expression of LPS with a different electrophoretic profile to that of wild-type RM7004. An unexpected feature of this mutation was that it appeared to influence the number of copies of 5'-CAAT-3' present in lic2A, a gene which is also required for biosynthesis and phase variable expression of the Gal alpha (1-4) beta Gal LPS epitope.
Assuntos
Elementos de DNA Transponíveis/genética , Genes Bacterianos/genética , Haemophilus influenzae/genética , Lipopolissacarídeos/biossíntese , Anticorpos Monoclonais , Proteínas de Bactérias/genética , Proteínas de Bactérias/imunologia , Proteínas de Bactérias/metabolismo , Técnicas Bacteriológicas , Sequência de Bases , Southern Blotting , Cromossomos Bacterianos/genética , DNA Bacteriano/genética , Elementos Facilitadores Genéticos/genética , Testes Genéticos , Haemophilus influenzae/citologia , Haemophilus influenzae/metabolismo , Dados de Sequência Molecular , Mutagênese/fisiologia , Plasmídeos , Reação em Cadeia da Polimerase , Mapeamento por Restrição , Análise de Sequência de DNA , Homologia de Sequência de AminoácidosRESUMO
The tetrameric repeat units 5'-CAAT-3' and 5'-GCAA-3' are associated with phase variable expression of lipopolysaccharide biosynthetic genes in Haemophilus influenzae. Four other tetrameric repeat units have also been reported from H. influenzae strain Rd, 5'-CAAC-3', 5'-GACA-3', 5'-AGCT-3', and 5'-TTTA-3', which are also associated with putative virulence factors. Using oligonucleotide probes corresponding to five tandem copies of each of these tetramers, we have screened three strains of Neisseria meningitidis and one each of Neisseria gonorrhoeae, Neisseria lactamica, Haemophilus parainfluenzae, Bordetella pertussis, Bordetella parapertussis, Bordetella bronchiceptica and Moraxella catarrhalis for the presence of these motifs. We have demonstrated the presence of multiple copies of the 5'-GCAA-3' motif in all the Neisseria strains tested, and also the repeated motif 5'-CAAC-3' in M. catarrhalis. We have further demonstrated by Southern blot analysis that the 5'-CAAC-3' repeats detected in M. catarrhalis are probably associated with the same genes as in H. influenzae, but that the 5'-GCAA-3' motifs in N. meningitidis are not. The use of characterised tetrameric DNA sequences as hybridisation probes may prove useful in the identification of novel phase variable virulence determinants in organisms other than H. influenzae.
Assuntos
Haemophilus/genética , Haemophilus/patogenicidade , Lipopolissacarídeos/biossíntese , Repetições de Microssatélites , Moraxella catarrhalis/genética , Moraxella catarrhalis/patogenicidade , Neisseria/genética , Neisseria/patogenicidade , Sequência de Bases , Primers do DNA/genética , DNA Bacteriano/genética , Expressão Gênica , Haemophilus/metabolismo , Humanos , Dados de Sequência Molecular , Moraxella catarrhalis/metabolismo , Neisseria/metabolismo , Sondas de Oligonucleotídeos/genética , Reação em Cadeia da Polimerase , Virulência/genéticaRESUMO
The availability of complete microbial genome sequences enormously facilitates experimental molecular investigations of the respective organisms by providing complete lists of genes, their genetic contexts, and their predicted functions. This can be used in a number of ways to focus studies on bacterial pathogenesis and also vaccine development (1,2). The complete genome sequences from two unrelated strains of Neisseria meningitidis, a derivative of isolate MC58 which originally expressed serogroup B capsule and strain Z2491, which is serogroup A, are now available (3,4). The genome sequences of both these strains were determined using the whole genome shotgun approach (5). In this approach, randomly sheared chromosomal DNA is cloned to make a small insert library (1.5-2.0 kb for MC58, 0.5-0.8 kb and 1.0-1.5 kb for Z2491), then each insert is sequenced from both ends using plasmidspecific primers. For the MC58 genome sequence, a large insert lambda library (8-24 kb) was also used. In the initial sequencing phase, 6-8 times coverage of the estimated size of the genome is generally achieved. The DNA sequences are linked together (assembled) into large contigs (a derivative of the word contiguous). Polymerase chain reaction (PCR) and sequencing of large insert libraries are then used to join the contigs, close gaps, and resolve ambiguities (see ref. 6 for a review).
RESUMO
In view of the importance of 5-N-acetyl neuraminic acid in bacterial pathogenesis, a sensitive, reproducible and reliable method for the determination of 5-N-acetyl neuraminic acid levels in lipopolysaccharide (LPS) is described and applied to 24 different non-typeable Haemophilus influenzae (NTHi) strains. The method involves analysis by high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD) of terminal 5-N-acetyl neuraminic acid residues released by neuraminidase treatment of O-deacylated LPS. The procedure is relatively fast and the instrumental effort is moderate. The results of the procedure were compared with data obtained by 1H NMR and electrospray ionisation-mass spectrometry (ESI-MS). The analysis of LPS from 24 NTHi strains showed that 5-N-acetyl neuraminic acid was found to be a common constituent of LPS in NTHi. Only one strain (NTHi 432) did not show any sialylation. Molar ratios (LPS/5-N-acetyl neuraminic acid) ranged between 5/1 and 500/1. Several strains in which no 5-N-acetyl neuraminic acid could be determined by other methods including 1H NMR and ESI-MS were shown to contain 5-N-acetyl neuraminic acid by this HPAEC-PAD procedure. The method was applied to determine levels of terminal 5-N-acetyl neuraminic acid in LPS from NTHi strains grown under different conditions and mutant strains containing inactive LPS biosynthetic genes.
Assuntos
Haemophilus influenzae/química , Lipopolissacarídeos/química , Ácido N-Acetilneuramínico/análise , Cromatografia por Troca Iônica/métodos , Cromatografia por Troca Iônica/normas , Haemophilus influenzae/classificação , Lipopolissacarídeos/metabolismo , Espectroscopia de Ressonância Magnética , Neuraminidase/metabolismo , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Espectrometria de Massas por Ionização por ElectrosprayRESUMO
The involvement of genes in the lic loci in H. influenzae LPS expression has been known for some time. However, it was not until recently that it was shown that the lic1 locus contains genes required for phase variable expression of phosphocholine substituents, while genes in the lic2 locus and lgtC are required for expression of the globoside trisaccharide, alpha-D-Galp-(1 --> 4)-beta-D-Galp-(1 --> 4)-beta-D-Glcp (i.e., the pK blood group epitope). The availability of the complete sequence of the H. influenzae strain Rd genome has facilitated significant progress in understanding the role of these and other genes in the expression and biosynthesis of LPS. We have employed a comparative structural fingerprinting strategy to establish the structural relationships among LPS from H. influenzae mutant strains in which putative biosynthesis genes were inactivated. Using this functional genomics approach, we have gained considerable insight into the genetic basis for intra-strain and strain-to-strain variation in epitope expression.
Assuntos
Haemophilus influenzae/genética , Lipopolissacarídeos/química , Sequência de Bases , Sequência de Carboidratos , DNA Bacteriano/genética , Expressão Gênica , Genes Bacterianos , Genômica , Haemophilus influenzae/classificação , Haemophilus influenzae tipo b/genética , Dados de Sequência Molecular , Estrutura Molecular , Mutação , Espectrometria de Massas por Ionização por ElectrosprayRESUMO
Simple sequence repeat (SSRs) of DNA are subject to high rates of mutation and are important mediators of adaptation in Haemophilus influenzae. Previous studies of the Rd KW20 genome identified the primacy of tetranucleotide SSRs in mediating phase variation (the rapid reversible switching of gene expression) of surface exposed structures such as lipopolysaccharide. The recent sequencing of the genomes of multiple strains of H. influenzae allowed the comparison of the SSRs (repeat units of one to nine nucleotides in length) in detail across four complete H. influenzae genomes and then comparison with a further 12 genomes when they became available. The SSR loci were broadly classified into three groups: (1) those that did not vary; (2) those for which some variation between strains was observed but this could not be linked to variation of gene expression; and (3) those that both varied and were located in regions consistent with mediating phase variable gene expression. Comparative analysis of 988 SSR associated loci confirmed that tetranucleotide repeats were the major mediators of phase variation and extended the repertoire of known tetranucleotide SSR loci by identifying ten previously uncharacterised tetranucleotide SSR loci with the potential to mediate phase variation which were unequally distributed across the H. influenzae pan-genome. Further, analysis of non-tetranucleotide SSR in the 16 strains revealed a number of mononucleotide, dinucleotide, pentanucleotide, heptanucleotide, and octanucleotide SSRs which were consistent with these tracts mediating phase variation. This study substantiates previous findings as to the important role that tetranucleotide SSRs play in H. influenzae biology. Two Brazilian isolates showed the most variation in their complement of SSRs suggesting the possibility of geographic and phenotypic influences on SSR distribution.
Assuntos
DNA Bacteriano/genética , Genoma Bacteriano/genética , Haemophilus influenzae/genética , Sequências Repetitivas de Ácido Nucleico/genética , DNA Bacteriano/metabolismo , Regulação Bacteriana da Expressão Gênica , Variação GenéticaRESUMO
The availability of complete genome sequences is a revolution in the study of microorganisms. A fully annotated genome sequence provides an interactive tool for scientists and influences the approach and focus of research. In this article I discuss the impact of genome sequencing projects of bacteria. Much useful data have been obtained but the experimental methods needed to fully exploit the information continue to develop. Some of the approaches and particular applications relevant to bacteria of clinical importance are discussed.
Assuntos
Bactérias/patogenicidade , Genoma Bacteriano , Bactérias/genética , Infecções Bacterianas/tratamento farmacológico , Vacinas Bacterianas , Genes Bacterianos , Haemophilus influenzae/genética , Serviços de Informação , Virulência/genéticaRESUMO
Biochemically deficient strains of Serratia marinorubra have been isolated with specific requirements for adenine, uracil, and threonine. Standard curves for dose to growth response have been obtained showing a linear sensitivity from 0.5 to 4.0 mg of adenine per liter of seawater, 0.1 to 2.0 mg of uracil per liter of seawater, and 0.5 to 10 mg of threonine per liter of seawater. These mutants have been used to test for the presence of their required metabolites in natural seawater samples from the Gulf of Mexico and adjacent bays. Of the three compounds under investigation, adenine was found in 10 samples, uracil in 2 samples, and threonine in none. The possible significance of these findings to the marine environment is discussed.
Assuntos
Adenina/análise , Técnicas Bacteriológicas , Serratia/metabolismo , Treonina/análise , Uracila/análise , Bioensaio , Mutação , Estações do Ano , Microbiologia da ÁguaRESUMO
The genomic relatedness among 36 strains of pink-pigmented facultatively methylotrophic bacteria (PPFMs) was estimated by determination of DNA base composition and by DNA:DNA hybridization studies. A reproducible hybridization system was developed for the rapid analysis of multiple DNA samples. Results indicated that the PPFMs comprise four major and several minor homology groups, and that they should remain grouped in a single genus, Methylobacterium.
Assuntos
DNA Bacteriano/classificação , Hibridização de Ácido Nucleico , Bactérias/classificação , Composição de Bases , Homologia de Sequência do Ácido NucleicoRESUMO
Lipopolysaccharide (LPS) is a major determinant of Neisseria, meningitidis virulence. A key feature of meningococcal LPS is the phase-variable expression of terminal structures which are proposed to have disparate roles in pathogenesis. In order to identify the biosynthetic genes for terminal LPS structures and the control mechanisms for their phase-variable expression, the lic2A gene, which is involved in LPS biosynthesis in Haemophilus influenzae, was used as a hybridization probe to identify a homologous gene in N. meningitidis strain MC58. The homologous region of DNA was cloned and nucleotide sequence analysis revealed three open reading frames (ORFs), two of which were homologous to the H. influenzae lic2A gene. All three ORFs were mutagenized by the insertion of antibiotic-resistance cassettes and the LPS from these mutant strains was analysed to determine if the genes had a role in LPS biosynthesis. Immunological and tricine-SDS-PAGE analysis of LPS from the mutant strains indicated that all three genes were probably transferases in the biosynthesis of the terminal lacto-N-neotetraose structure of meningococcal LPS. The first ORF of the locus contains a homopolymeric tract of 14 guanosine residues within the 5'-end of the coding sequence. As the lacto-N-neotetraose structure in meningococcal LPS is subject to phase-variable expression, colonies that no longer expressed the terminal structure, as determined by monoclonal antibody binding, were isolated. Analysis of an 'off' phase variant revealed a change in the number of guanosine residues resulting in a frameshift mutation, indicating that a slipped-strand mispairing mechanism, operating in the first ORF, controls the phase-variable expression of lacto-N-neotetraose.
Assuntos
Lipopolissacarídeos/biossíntese , Neisseria meningitidis/genética , Oligossacarídeos/genética , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Southern Blotting , Clonagem Molecular , Eletroforese em Gel de Poliacrilamida , Mutação da Fase de Leitura , Regulação Bacteriana da Expressão Gênica , Genes Bacterianos , Lipopolissacarídeos/química , Lipopolissacarídeos/imunologia , Dados de Sequência Molecular , Mutagênese Insercional , Neisseria meningitidis/patogenicidade , Fases de Leitura Aberta , Plasmídeos , Recombinação Genética , Mapeamento por Restrição , Transferases/genéticaRESUMO
Transformation of tryptophan auxotrophs of Streptomyces coelicolor A3(2) and subsequent analysis have allowed the identification of four tryptophan biosynthetic genes. Subcloning, complementation of trp strains, nucleotide sequencing of 5.1 kb and 1.95 kb of DNA and subsequent homology comparisons identified the trpC, trpB and trpA genes and trpD gene respectively. The arrangement of genes in the trpCBA cluster is unusual in that trpC is separated by a small open reading frame, trpX, from the potentially translationally coupled trpB and trpA genes. Sequence analysis of the trpD gene revealed the presence of a large mRNA loop structure directly upstream of the trpD-coding region. S1 nuclease mapping studies of trpCXBA have revealed two major potential transcription start points, one just upstream of the trpC gene and the other located upstream of the trpX gene. S1 nuclease mapping of the trpD region revealed four fragment end-points. Quantitative S1 nuclease protection assays and a promoterless catechol dioxygenase reporter gene have revealed that the expression of all these genes is growth phase dependent and growth rate dependent, expression being maximal during early exponential phase and dropping off sharply in late exponential phase. This growth phase-dependent and growth rate-dependent regulation is the first reported in streptomycete primary metabolism.
Assuntos
Dioxigenases , Regulação Bacteriana da Expressão Gênica/genética , Genes Bacterianos , Streptomyces/genética , Triptofano/biossíntese , Sequência de Aminoácidos , Sequência de Bases , Catecol 1,2-Dioxigenase , Clonagem Molecular , Genes Reporter , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Oxigenases/genética , Regiões Promotoras Genéticas , RNA Mensageiro/genética , Mapeamento por Restrição , Análise de Sequência , Endonucleases Específicas para DNA e RNA de Cadeia Simples/metabolismo , Streptomyces/crescimento & desenvolvimento , Transcrição Gênica , Triptofano/genéticaRESUMO
We describe an integrated system for the analysis of DNA sequence motifs within complete bacterial genome sequences. This system is based around ACeDB, a genome database with an integrated graphical user interface; we identify and display motifs in the context of genetic, sequence and bibliographic data. Tomb et aL (1997) previously reported the identification of contingency genes in Helicobacter pylori through their association with homopolymeric tracts and dinucleotide repeats. With this as a starting point, we validated the system by a search for this type of repeat and used the contextual information to assess the likelihood that they mediate phase variation in the associated open reading frames (ORFs). We found all of the repeats previously described, and identified 27 putative phase-variable genes (including 17 previously described). These could be divided into three groups: lipopolysaccharide (LPS) biosynthesis, cell-surface-associated proteins and DNA restriction/modification systems. Five of the putative genes did not have obvious homologues in any of the public domain sequence databases. The reading frame of some ORFs was disrupted by the presence of the repeats, including the alpha(1-2) fucosyltransferase gene, necessary for the synthesis of the Lewis Y epitope. An additional benefit of this approach is that the results of each search can be analysed further and compared with those from other genomes. This revealed that H. pylori has an unusually high frequency of homopurine:homopyrimidine repeats suggesting mechanistic biases that favour their presence and instability.