Rosemary J. Redfield

Is quorum sensing a side effect of diffusion sensing

Many bacteria appear to communicate by releasing and sensing autoinducer molecules, which are believed to function primarily as sensors of population density. However, this quorum-sensing hypothesis rests on very weak foundations, as neither the need for group action nor the selective conditions required for its evolution have been demonstrated. Here, I argue for a more direct function of autoinducer secretion and response - the ability to determine whether secreted molecules rapidly move away from the cell. This diffusion sensing allows cells to regulate secretion of degradative enzymes and other effectors to minimize losses owing to extracellular diffusion and mixing.

Do bacteria have sex

Do bacteria have genes for genetic exchange? The idea that the bacterial processes that cause genetic exchange exist because of natural selection for this process is shared by almost all microbiologists and population geneticists. However, this assumption has been perpetuated by generations of biology, microbiology and genetics textbooks without ever being critically examined.

Natural Transformation and DNA Uptake Signal Sequences in Actinobacillus actinomycetemcomitans

Actinobacillus actinomycetemcomitans is a member of the family Pasteurellaceae and a major causative agent of periodontitis. While several genera from this family are known to be competent for transformation, A. actinomycetemcomitans has yet to be fully characterized. Here we show that the competence of A. actinomycetemcomitans is remarkably similar to that of Haemophilus influenzae. In addition to having a similar frequency of transformation as H. influenzae, A. actinomycetemcomitans competence could also be induced at least 100-fold by cyclic AMP, suggesting that, as in H. influenzae, at least some competence genes are regulated by catabolite repression. Even more intriguing was the discovery of a putative A. actinomycetemcomitans DNA uptake signal sequence (USS) virtually identical to the USS of H. influenzae. Moreover, we provide evidence that this sequence functions in the same capacity as that from H. influenzae; the sequence appears to be required and sufficient for DNA uptake in a variety of assays. Finally, we have taken advantage of this system to develop a simple, highly efficient competence-based method for generating site-directed mutations in the wild-type fimbriated A. actinomycetemcomitans.

Absence of Detectable Arsenate in DNA from Arsenate-Grown GFAJ-1 Cells

Resisting Arsenic The discovery of a bacterium living in the extreme conditions of Mono Lake, California, created a major controversy because it was claimed to be able to grow solely on arsenic and could substitute arsenate for phosphate in its key macromolecules, including DNA. Working with the same Halomonas spp. bacterium, known as GFAJ-1, and ultrapure reagents, Erb et al. (p. 467) found that the bacterium needed a low level of phosphate (1.6 µM) to grow at all. Rather than significant specific arsenic incorporation, when the organism was grown in 40 mM arsenic, its nucleic acids acquired a trace of arsenic. Similarly, Reaves et al. (p. 470) found that GFAJ-1 could not grow in the absence of phosphate and, moreover, that its growth was not stimulated by the addition of arsenate, although a trace amount of arsenic was also detected in DNA. Thus, GFAJ-1 shows no particular facility to substitute arsenic for phosphate, when phosphate is limiting, but it can tolerate high concentrations of the poison while efficiently scavenging phosphate. Claims of arsenic substitution for phosphorus in the biomolecules of a Mono Lake bacterium are not independently reproduced. A strain of Halomonas bacteria, GFAJ-1, has been claimed to be able to use arsenate as a nutrient when phosphate is limiting and to specifically incorporate arsenic into its DNA in place of phosphorus. However, we have found that arsenate does not contribute to growth of GFAJ-1 when phosphate is limiting and that DNA purified from cells grown with limiting phosphate and abundant arsenate does not exhibit the spontaneous hydrolysis expected of arsenate ester bonds. Furthermore, mass spectrometry showed that this DNA contains only trace amounts of free arsenate and no detectable covalently bound arsenate.

Non-canonical CRP sites control competence regulons in Escherichia coli and many other γ-proteobacteria

Escherichia colis cAMP receptor protein (CRP), the archetypal bacterial transcription factor, regulates over a hundred promoters by binding 22 bp symmetrical sites with the consensus core half-site TGTGA. However, Haemophilus influenzae has two types of CRP sites, one like E.colis and one with the core sequence TGCGA that regulates genes required for DNA uptake (natural competence). Only the latter ‘CRP-S’ sites require both CRP and the coregulator Sxy for activation. To our knowledge, the TGTGA and TGCGA motifs are the first example of one transcription factor having two distinct binding-site motifs. Here we show that CRP-S promoters are widespread in the γ-proteobacteria and demonstrate their Sxy-dependence in E.coli. Orthologs of most H.influenzae CRP-S-regulated genes are ubiquitous in the five best-studied γ-proteobacteria families, Enterobacteriaceae, Pasteurellaceae, Pseudomonadaceae, Vibrionaceae and Xanthomonadaceae. Phylogenetic footprinting identified CRP-S sites in the promoter regions of the Enterobacteriaceae, Pasteurellaceae and Vibrionaceae orthologs, and canonical CRP sites in orthologs of genes known to be Sxy-independent in H.influenzae. Bandshift experiments confirmed that E.coli CRP-S sequences are low affinity binding sites for CRP, and mRNA analysis showed that they require CRP, cAMP (CRPs allosteric effector) and Sxy for gene induction. This work suggests not only that the γ-proteobacteria share a common DNA uptake mechanism, but also that, in the three best studied families, their competence regulons share both CRP-S specificity and Sxy dependence.

Transformation of Haemophilus influenzae

Conjugation: Although H. influenzae genes may be transferred by plasmid-mediated conjugation, this process is not discussed further below as it has received little attention and is rarely used for strain construction. Conjugative plasmids are common in H. influenzae as in other bacteria [1, 2, 3]. The F plasmid of Escherichia coli can conjugate into H. influenzae cells [4] (and into many other cell types), and can then transfer by conjugation from one H. influenzae strain to another. As in E. coli, efficient transfer of chromosomal genes requires a region of homology between the plasmid and the chromosome. A method for F-mediated conjugation into H. influenzae is described by Deich and Green[4].

Regulation of competence development and sugar utilization in Haemophilus influenzae Rd by a phosphoenolpyruvate:fructose phosphotransferase system

Changes in intracellular cAMP concentration play important roles in Haemophilus influenzae, regulating both sugar utilization and competence for natural transformation. In enteric bacteria, cAMP levels are controlled by the phosphoenolpyruvate:glycose phosphotransferase system (PTS) in response to changes in availability of the preferred sugars it transports. We have demonstrated the existence of a simple PTS in H. influenzae by several methods. We have cloned the H. influenzaeptsI gene, encoding PTS Enzyme I; genome analysis locates it in a pts operon structurally homologous to those of enteric bacteria. In vitro phosphorylation assays confirmed the presence of functional PTS components. A ptsI null mutation reduced fructose uptake to 1% of the wild‐type rate, and abolished fructose fermentation even when exogenous cAMP was provided. The ptsI mutation also prevented fermentation of ribose and galactose, but utilization of these cAMP‐dependent sugars was restored by addition of cAMP. In wild‐type cells the non‐metabolizable fructose analogue xylitol prevented fermentation of these sugars, confirming that the fructose PTS regulates cAMP levels. Development of competence under standard inducing conditions was reduced 250‐fold by the ptsI mutation, unless cells were provided with exogenous cAMP. Competence is thus shown to be under direct nutritional control by a fructose‐specific PTS.

Competence development by Haemophilus influenzae is regulated by the availability of nucleic acid precursors

DNA uptake by naturally competent bacteria provides cells with both genetic information and nucleotides. In Haemophilus influenzae, competence development requires both cAMP and an unidentified signal arising under starvation conditions. To investigate this signal, competence induction was examined in media supplemented with nucleic acid precursors. The addition of physiological levels of AMP and GMP reduced competence 200‐fold and prevented the normal competence‐induced transcription of the essential competence genes comA and rec‐2. The rich medium normally used for growth allows only limited competence. Capillary electrophoresis revealed only a subinhibitory amount of AMP and no detectable GMP, and the addition of AMP or GMP to this medium also reduced competence 20‐ to 100‐fold. Neither a functional stringent response system nor a functional phosphoenolpyruvate:glycose phosphotransferase system (PTS) was found to be required for purine‐mediated repression. Added cAMP partially restored both transcription of competence genes and competence development, suggesting that purines may reduce the response to cAMP. Potential binding sites for the PurR repressor were identified in several competence genes, suggesting that competence is part of the PUR regulon. These observations are consistent with models of competence regulation, in which depleted purine pools signal the need for nucleotides, and support the hypothesis that competence evolved primarily for nucleotide acquisition.

EXTENSIVE VARIATION IN NATURAL COMPETENCE IN HAEMOPHILUS INFLUENZAE

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.

Phylogenetic placement of Trichonympha

Flagellated protists of the Class Hypermastigida have previously been classified on morphology alone, since no molecular sequences have been available. We have isolated DNA from 350 cells of the hypermastigote Trichonympha, manually collected from the hindgut of Zootermopsis angusticollis, and used this DNA as template for polymerase chain reaction amplification of the small‐subunit ribosomal RNA gene. The DNA sequence of the amplified fragment is closely related to that of a previously‐unidentified gut symbiont from the termite Reticulitermes flavipes, and phylogenetic analysis places both sequences as a sister group to the known trichomonads. in agreement with the morphological classification.