Molecular Microbiology

Olsson, Jan; Dasgupta, Santanu; Berg, Otto G.; Nordström, Kurt

doi: 10.1046/j.1365-2958.2002.02954.xpmid: 12067334

Summary The classical Meselson–Stahl density shift experiment was used to determine the length of the eclipse period in Escherichia coli, the minimum time period during which no new initiation is allowed from a newly replicated origin of chromosome replication, oriC. Populations of bacteria growing exponentially in heavy (15NH4+ and 13C6‐glucose) medium were shifted to light (14NH4+ and 12C6‐glucose) medium. The HH‐, HL‐ and LL‐DNA were separated by CsCl density gradient centrifugation, and their relative amounts were determined using radioactive gene‐specific probes. The eclipse period, estimated from the kinetics of conversion of HH‐DNA to HL‐ and LL‐DNA, turned out to be 0.60 generation times for the wild‐type strain. This was invariable for widely varying doubling times (35, 68 and 112 min) and was independent of the chromosome locus at which the eclipse period was measured. For strains with seqA, dam and damseqA mutants, the length of the eclipse period was 0.16, 0.40 and 0.32 generation times respectively. Thus, initiations from oriC were repressed for a considerable proportion of the generation time even when the sequestration function seemed to be severely compromised. The causal relationship between the length of the eclipse period and the synchrony of initiations from oriC is discussed.

Martin, Robert G.; Rosner, Judah L.

doi: 10.1046/j.1365-2958.2002.02985.xpmid: 12067348

Summary Microarray analyses are providing a plethora of data concerning transcriptional responses to specific gene regulators and their inducers but do not distinguish between direct and indirect responses. Here, we identify directly activated promoters of the overlapping marA, soxS and rob regulon(s) of Escherichia coli by applying informatics, genomics and molecular genetics to microarray data obtained by others. Those studies found that overexpression of marA, or the treatment of cells with salicylate to derepress marA, or treatment with paraquat to induce soxS, resulted in elevated transcription of 153 genes. However, only 27 out of the promoters showed increased transcription under at least two of the aforementioned conditions and eight of those were previously known to be directly activated. A computer algorithm was used to identify potential activator binding sites located upstream of the remaining 19 promoters of this subset, and conventional genetic and biochemical approaches were applied to test whether these sites are critical for activation by the homologous MarA, SoxS and Rob transcriptional activators. Only seven out of the 19 promoters were found to be activated when fused to lacZ and tested as single lysogens. All seven contained an essential activator binding site. The remaining promoters were insensitive to stimulation by the inducers suggesting that the great majority of elevated microarray transcripts either were misidentified or resulted from indirect effects requiring sequences outside of the promoter region. We estimate that the total number of directly activated promoters in the regulon is less than 40.

Claessen, Dennis; Wösten, Han A. B.; Keulen, Geertje van; Faber, Onno G.; Alves, Alexandra M. C. R.; Meijer, Wim G.; Dijkhuizen, Lubbert

doi: 10.1046/j.1365-2958.2002.02980.xpmid: 12067338

Summary The filamentous bacteria Streptomyces coelicolor and Streptomyces lividans exhibit a complex life cycle. After a branched submerged mycelium has been established, aerial hyphae are formed that may septate to form chains of spores. The aerial structures possess several surface layers of unknown nature that make them hydrophobic, one of which is the rodlet layer. We have identified two homologous proteins, RdlA and RdlB, that are involved in the formation of the rodlet layer in both streptomycetes. The rdl genes are expressed in growing aerial hyphae but not in spores. Immunolocalization showed that RdlA and RdlB are present at surfaces of aerial structures, where they form a highly insoluble layer. Disruption of both rdlA and rdlB in S. coelicolor and S. lividans (ΔrdlAB strains) did not affect the formation and differentiation of aerial hyphae. However, the characteristic rodlet layer was absent. Genes rdlA and rdlB were also expressed in submerged hyphae that were in contact with a hydrophobic solid. Attachment to this substratum was greatly reduced in the ΔrdlAB strains. Sequences homologous to rdlA and rdlB occur in a number of streptomycetes representing the phylogenetic diversity of this group of bacteria, indicating a general role for these proteins in rodlet formation and attachment.

Tauschek, Marija; Strugnell, Richard A.; Robins‐Browne, Roy M.

doi: 10.1046/j.1365-2958.2002.02968.xpmid: 12067342

Summary We have characterized the LEE pathogenicity islands (PAIs) of two rabbit‐specific strains of enteropathogenic E. coli (REPEC), 83/39 (serotype O15:H‐) and 84/110‐1 (O103:H2), and have compared them to homologous loci from the human enteropathogenic and enterohaemorrhagic E. coli strains, E2348/69 and EDL933, and another REPEC strain, RDEC‐1. All five PAIs contain a 34 kb core region that is highly conserved in gene order and nucleotide sequence. However, the LEE of 83/39 is significantly larger (59 540 basepairs) than those of the human strains, which are less than 44 kb, and has inserted into pheU tRNA. The regions flanking the 34 kb core of 83/39 contain homologues of two putative virulence determinants, efa1/lifA and senA. The LEE of 84/110‐1 is approximately 85 kb and is located at pheV tRNA. Its core is almost identical to those of 83/39 and RDEC‐1, apart from a larger espF gene, but its flanking regions contain trcA, a putative virulence determinant of EPEC. All three REPEC LEE PAIs contain a gene for an integrase, Int‐phe. The LEE PAI of 84/110‐1 is also flanked by short direct repeats (representing the 3′‐end of pheV tRNA), suggesting that it may be unstable. To investigate this possibility, we constructed a LEE::sacB derivative of 84/110‐1 and showed that the PAI was capable of spontaneous deletion. We also showed that Int‐phe can mediate site‐specific integration of foreign DNA at the pheU tRNA locus of E. coli DH1. Together these results indicate possible mechanisms of mobilization and integration of the LEE PAI.

Weissman, Ziva; Shemer, Revital; Kornitzer, Daniel

doi: 10.1046/j.1365-2958.2002.02976.xpmid: 12067343

Summary Efficient iron acquisition is an essential requirement for growth of pathogenic organisms in the iron‐poor host environment. In Saccharomyces cerevisiae, high‐affinity iron import depends on the multicopper ferroxidase ScFet3. ScFet3 biogenesis in the trans‐Golgi compartment requires a copper‐transporting P‐type ATPase, ScCcc2. Here, we describe the isolation by functional complementation of a Ccc2 homologue from the pathogenic yeast Candida albicans. CaCcc2 is functionally distinct from a previously described C. albicans copper‐transporting P‐type ATPase, CaCrp1, which appears to be specifically involved in copper detoxification. Regulation of CaCCC2 and the phenotype of the homozygous CaCCC2 deletion indicate that it is required for high‐affinity iron import, making it the bona fide CCC2 homologue of C. albicans. Remarkably, in a mouse model of systemic infection, the Caccc2Δ strain displayed robust proliferation and no significant reduction in pathogenicity, suggesting the existence of alternative mechanisms of iron uptake from host tissues. We identify haemin and haemoglobin as potential iron sources that can be used by C. albicans in a CaCcc2‐independent manner.

Minogue, Timothy D.; Trebra, Markus Wehland‐von; Bernhard, Frank; Bodman, Susanne B. von

doi: 10.1046/j.1365-2958.2002.02987.xpmid: 12067349

Summary Capsular polysaccharide synthesis and virulence in the plant pathogenic bacterium Pantoea stewartii ssp. stewartii requires the quorum‐sensing regulatory proteins, EsaR and EsaI, and the diffusible inducer N‐(3‐oxo‐hexanoyl)‐L‐homoserine lactone. Prior mu‐tational studies suggested that EsaR might function as a repressor of quorum sensing in the control of capsular polysaccharide synthesis. Further, a lux box‐like palindromic sequence coinciding with the putative –10 element of the esaR promoter suggested a possible negative autoregulatory role for EsaR. This report presents genetic evidence that EsaR represses the esaR gene under inducer‐limiting conditions, and that addition of inducer promotes rapid, dose‐dependent derepression. DNA mobility‐shift assays and analyses by surface plasmon resonance refractometry show that EsaR binds target DNAs in a ligand‐free state, and that inducer alters the binding characteristics of EsaR. Physical measurements indicate that the EsaR protein binds N‐(3‐oxo‐hexanoyl)‐L‐homoserine lactone, in a 1:1 protein:ligand ratio, and that inducer binding enhances the thermal stability of the EsaR protein. These combined genetic and biochemical data establish that EsaR regulates its own expression by signal‐independent repression and signal‐dependent derepression. Additionally, we provide evidence that EsaR does not govern the expression of the linked esaI gene, thus EsaR has no role in controlling coinducer synthesis.

Ansaldi, Mireille; Marolt, Darja; Stebe, Tina; Mandic‐Mulec, Ines; Dubnau, David

doi: 10.1046/j.1365-2958.2002.02977.xpmid: 12067344

Summary Natural genetic competence in Bacillus subtilis is controlled by quorum‐sensing (QS). The ComP– ComA two‐component system detects the signalling molecule ComX, and this signal is transduced by a conserved phosphotransfer mechanism. ComX is synthesized as an inactive precursor and is then cleaved and modified by ComQ before export to the extracellular environment. The comQXP′ loci of a set of natural Bacillus isolates have been sequenced and shown to possess a striking polymorphism that determines specific patterns of both activation and inhibition of the quorum‐sensing response. We have developed a simple purification method for the modified peptide signalling pheromones allowing the characterization of four distinct ComX molecules representing different pherotypes. Genetic and biochemical evidence demonstrate that all the ComX variants are isoprenylated by the post‐translational modification of a conserved tryptophan residue and that the modifications on the ComX peptide backbones vary in mass among the various pherotypes. These results give new insights into peptidemediated quorum‐sensing signalling in Gram‐positive bacteria and emphasize the role of isoprenylation in bacterial signal transduction.

Wang, Hong‐Liang; Postier, Bradley L.; Burnap, Robert L.

doi: 10.1046/j.1365-2958.2002.02983.xpmid: 12067339

Summary Primary ion pumps and antiporters exist as multigene families in the Synechocystis sp. PCC 6803 genome and show very strong homologies to those found in higher plants. The gene knock‐outs of five putative Na+/H+ antiporters (slr1727, sll0273, sll0689, slr1595 and slr0415) and seven cation ATPases (sll1614, sll1920, slr0671–72, slr0822, slr1507–08–09, slr1728– 29 and slr1950) in the model cyanobacterium (http://www.kazusa.or.jp/cyano/cyano.html) were performed in this study relying on homologous recombination with mutagenenic fragments constructed using a fusion polymerase chain reaction (PCR) approach. The impacts of these gene knock‐outs were evaluated in terms of Na+ and pH, and light‐induced acidification and alkalization that are asso‐ciated with inorganic carbon uptake. Two of the five putative antiporter mutants exhibit a characteristic interplay between the pH and Na+ dependence of growth, but only one of the antiporters appears to be necessary for high NaCl tolerance. On the other hand, the mutation of one of the two copper‐trafficking ATPases produces a cell line that shows acute NaCl sensitivity. Additionally, disruptions of a putative Ca2+‐ATPase and a gene cluster encoding a putative Na+‐ATPase subunit also cause high NaCl sensitivity. The findings and possible mechanisms are discussed in relation to the potential roles of these transporters in Synechocystis sp. PCC 6803.

Eksi, Saliha; Stump, Amy; Fanning, Sarah L.; Shenouda, Mary I.; Fujioka, Hisashi; Williamson, Kim C.

doi: 10.1046/j.1365-2958.2002.02986.xpmid: 12067340

Summary For malaria to be transmitted, the Plasmodium falciparum parasite must invade an erythrocyte and undergo gametocytogenesis. When mature intraerythrocytic gametocytes are taken up in a blood meal by a mosquito they emerge as gametes and, once fertilized, continue to differentiate into infectious sporozoites. One of the major proteins associated with the surface of the parasite during gamete differentiation is Pfs230, a 360 kDa member of a family of P. falciparum proteins that contains a repeated cysteine motif domain. To characterize the role of different regions of Pfs230, the gene was disrupted by targeted integration and clones isolated that expressed distinct sections of Pfs230. Independent clones D1.356 a and b express the first 452 amino acids (aa) of Pfs230 and do not contain a cysteine motif domain, whereas clones D2.850 a and b express the first 950 aa, including the first cysteine motif domain. Although both sets of clones undergo gametogenesis and produce morphologically normal gametes, neither truncated Pfs230 is located on the surface of the gamete. In clones D1.356 a and b, the 452 aa Pfs230 is secreted into the parasitophorous vacuole and released as a soluble protein when the parasite emerges from the erythrocyte as a gamete. In marked contrast, the 950 aa form of Pfs230 expressed by clones D2.850 a and b is sequestered in a novel tubular compartment in the erythrocyte cytoplasm. This sexual‐stage tubular intraerythrocytic compartment (STIC) is not recognized by antibodies specific for proteins associated with the parasitophorous vacuole membrane (Pfs16 or Exp‐1) or Maurer’s clefts (Pfsbp 1 or mAb LWL1) or intraerythrocytic asexual parasite proteins (PfEMP2 or HRP II).

Voncken, Frank; Boxma, Brigitte; Tjaden, Joachim; Akhmanova, Anna; Huynen, Martijn; Verbeek, Fons; Tielens, Aloysius G. M.; Haferkamp, Ilka; Neuhaus, H. Ekkehard; Vogels, Godfried; Veenhuis, Marten; Hackstein, Johannes H. P.