Defined Nongrowth Media for Stage II Development of Competence in
            <i>Haemophilus influenzae</i>

Herriott, Roger M.; Meyer, Elisabeth; Vogt, Marcella

doi:10.1128/jb.101.2.517-524.1970

Cited by 384 publications

(174 citation statements)

References 25 publications

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“…A 5-mL overnight culture, started from a frozen stock, was diluted 1:100 into fresh sBHI (25 mL in a 250-mL flask). Once cells were growing exponentially (OD 600 0.1-0.4), they were collected by filtration, washed in MIV competence medium (Herriott et al 1970;Poje and Redfield 2003) and then transferred to an equal volume of MIV and returned to the shaker for 100 min. Cells were then either immediately incubated with DNA or mixed with glycerol (16% final concentration) and frozen at −80 • C for assay at a later date.…”

Section: Competence Induction With Starvation Mediummentioning

confidence: 99%

“…However, because DNA uptake assays are much less sensitive than transformation assays, uptake is best measured when competence has been fully induced. In strain Rd this is accomplished by transferring exponentially growing cells to MIV, a starvation medium containing amino acids but no nucleotide precursors or essential cofactors (Herriott et al 1970). As this method has been successfully used to induce competence in other strains (e.g., see Swords et al 2000;Bakaletz et al 2005), we used it to prepare all strains for DNA uptake assays.…”

Section: Differences In Dna Uptakementioning

confidence: 99%

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EXTENSIVE VARIATION IN NATURAL COMPETENCE INHAEMOPHILUS INFLUENZAE

Maughan

Redfield

2009

Evolution

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The ability of some bacteria to take up and recombine DNA from the environment is an important evolutionary problem because its function is controversial; although populations may benefit in the long-term from the introduction of new alleles, cells also reap immediate benefits from the contribution of DNA to metabolism. To clarify how selection has acted, we have characterized competence in natural isolates of H. influenzae by measuring DNA uptake and transformation. Most of the 34 strains we tested became competent, but the amounts of DNA they took up and recombined varied more than 1000-fold. Differences in recombination were not due to sequence divergence and were only partly explained by differences in the amounts of DNA taken up. One strain was highly competent during log phase growth, unlike the reference strain Rd, but several strains did not develop competence under any of the tested conditions. Analysis of competence genes identified genetic defects in two poorly transformable strains.These results show that strains can differ considerably in the amount of DNA they take up and recombine, indicating that the benefit associated with competence is likely to vary in space and/or time. K E Y W O R D S :Competence, DNA uptake, polymorphism, selection-natural, transformation, variation.Natural competence is a regulated physiological state in which bacterial cells actively transport DNA from their external environment into the cytoplasm. (This differs from artificial competence that uses electrical or chemical treatments to passively permeabilize cellular membranes.) The evolutionary function of this genetically programmed ability remains controversial because DNA taken up by cells has three potential benefits, as a food source, as a template for DNA repair, and as a source of new alleles. First, the cell's pool of nucleotides is enriched by degradation of one DNA strand in the cytoplasm, by uptake of nucleotides that have been released by degradation of the other DNA strand at the cell surface, and by degradation of any chromosomal DNA strands displaced by homologous recombination. Second, the internalized DNA strand may be used as a template for recombinational repair of a homologous strand containing otherwise-irreparable DNA damage. Third, the genotype of the cell may change if recombination introduces a different allele ("transformation").

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Section: Competence Induction With Starvation Mediummentioning

confidence: 99%

Section: Differences In Dna Uptakementioning

confidence: 99%

EXTENSIVE VARIATION IN NATURAL COMPETENCE INHAEMOPHILUS INFLUENZAE

Maughan

Redfield

2009

Evolution

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“…Arginine (group A) has been already described as an essential amino acid for Haemophilus influenzae growth. 12,13,56,57 Other amino acids have been shown to be important for certain strains, as is the case of isoleucine and valine for FID strain and isoleucine, leucine and tyrosine for RdTr strain. In the case of the Rd strain, there is no essential amino acid for growth.…”

Section: Discussionmentioning

confidence: 99%

“…9,10 Defined media were also tested but they were not feasible for industrial purposes due to low biomass and PRP production. [11][12][13] Hib has been exhaustively studied from an epidemiologic and microbiological point of view since the first polysaccharide vaccine was produced. However, there is little information available on the improvements of an industrial production process and when available, process conditions are protected by patents.…”

mentioning

confidence: 99%

Experimental design and metabolic flux analysis tools to optimize industrially relevant Haemophilus influenzae type b growth medium

Silva

Portela

Albani

et al. 2017

Biotechnology Progress

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Haemophilus influenzae type b (Hib), a Gram-negative capsulated bacterium, is a causative agent of meningitis worldwide. The capsular polysaccharide, a high molecular mass polymer consisting of the repeated units of the polyribosyl-ribitol-phosphate, is considered the main virulence factor and it is used as an antigen to vaccines, conjugated to a carrier protein. The industrial production of the polysaccharide requires the cultivation of Hib in rich medium, which impacts process costs and product recovery. In this study, a central composite rotational experimental design strategy was used to access the influence of key components of culture medium (soy peptone, yeast extract and glucose) on biomass formation and polysaccharide production in shake-flasks. The optimized medium formulation, containing half of the usual yeast extract and soytone concentrations, was further validated in batch bioreactor cultivations. High polysaccharide production (∼500 mg/L) was obtained in a cheaper and more competitive production process for use in Hib vaccine production. In addition, simulations of a metabolic model describing Hib central metabolism were used to assess the role of key amino acids on growth. A chemically defined medium supplemented only with amino acids from α-ketoglutarate and oxaloacetate families as well as phenylalanine was suggested as a promising alternative for reduced acetate accumulation and enhanced polysaccharide production in Hib cultures. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:1508-1519, 2017.

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“…The chloramphenicol resistance cassette from plasmid pACYC184 (New England BioLabs, Beverly, MA) was inserted between the upstream and downstream fragments in the plasmid. Haemophilus influenzae strain Rd was transformed using the M IV method (Herriott et al, 1970). A chloramphenicol-resistant transformant was obtained.…”

Section: Construction Of a P2-deficient Mutantmentioning

confidence: 99%

Antibodies directed at a conserved motif in loop 6 of outer membrane protein P2 of nontypeableHaemophilus influenzaerecognize multiple strains in immunoassays

Neary¹,

Murphy

2006

FEMS Immunology &Amp; Medical Microbiology

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The P2 porin is the most abundant protein in the outer membrane of nontypeable Haemophilus influenzae. Analysis of P2 sequences from a limited number of strains reveals the presence of both heterogeneous and conserved surface-exposed loops of the P2 molecule among strains. We have previously shown that antibodies raised against the loop 6 sequence of P2 from strain 5657 are bactericidal against multiple isolates. In this study, we determined the nucleotide sequence of the loop 6 region of the P2 molecule from 108 strains of nontypeable H. influenzae in order to assess more rigorously the degree of conservation of loop 6. Based on this analysis, we identified a conserved sequence, different from that of strain 5657, that occurs in approximately one-third of the strains sequenced. To assess the potential of this peptide as a vaccine antigen, antibodies raised to a multiple antigenic peptide corresponding to this sequence were characterized with respect to specificity for the P2 molecule and reactivity with heterologous strains in immunoblot assay, flow cytometry and bactericidal assays. Antibodies were reactive to the P2 molecule of 16 of 20 strains tested by immunoblot assay. Antibodies recognized nine of the 20 strains in a flow cytometry assay, and 13 of 20 demonstrated complement-mediated killing in bactericidal assays. These results support the concept of using conserved regions of the P2 protein as a vaccine antigen.

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Defined Nongrowth Media for Stage II Development of Competence in Haemophilus influenzae

Cited by 384 publications

References 25 publications

EXTENSIVE VARIATION IN NATURAL COMPETENCE INHAEMOPHILUS INFLUENZAE

EXTENSIVE VARIATION IN NATURAL COMPETENCE INHAEMOPHILUS INFLUENZAE

Experimental design and metabolic flux analysis tools to optimize industrially relevant Haemophilus influenzae type b growth medium

Antibodies directed at a conserved motif in loop 6 of outer membrane protein P2 of nontypeableHaemophilus influenzaerecognize multiple strains in immunoassays

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