Molecular Microbiology

Carlson, Karin; Kosturko, Linda D.; Nyström, Anna‐Chey

doi: 10.1046/j.1365-2958.1999.01281.xpmid: 10200960

The 136 codon (408 bp) denA gene encoding endonuclease II (EndoII) of bacteriophage T4 was unambiguously identified through sequencing and subsequent cloning. EndoII prepared from cloned DNA through coupled in vitro transcription–translation nicked and cut DNA in vitro in a sequence‐specific manner. In vitro (and in vivo ), the bottom strand was nicked between the first and second base pair to the right of a top‐strand CCGC motif shared by favoured in vitro and in vivo cleavage sites; top‐strand cleavage positions varied. To the right of the cleavage position, favoured in vitro sites lacked a sequence element conserved at favoured in vivo sites. In pBR322 DNA, the sites cleaved in vivo as previously described were also cleaved in vitro, but in vitro additional sites were nicked or cleaved and the preference for individual sites was different. Also, different from the in vivo reaction, nicking was more frequent than ds cutting; in many copies of a ds cleavage site, only the bottom strand was nicked in vitro. A model is discussed in which sequential nicking of the two strands, and different factors influencing bottom‐strand nicking and top‐strand nicking, can explain the differences between the in vitro and the in vivo reaction.

Arigoni, Fabrizio; Guérout‐Fleury, Anne‐Marie; Barák, Imrich; Stragier, Patrick

doi: 10.1046/j.1365-2958.1999.01282.xpmid: 10200961

Making a spore in Bacillus subtilis requires the formation of two cells, the forespore and the mother cell, which follow dissimilar patterns of gene expression. Cell specificity is first established in the forespore under the control of the σF factor, which is itself activated through the action of the SpoIIE serine phosphatase, an enzyme targeted to the septum between the two cells. Deletion of the 10 transmembrane segments of the SpoIIE protein leads to random distribution of SpoIIE in the cytoplasm. Activation of σF is slightly delayed and less efficient than in wild type, but it remains restricted to the forespore in a large proportion of cells and the bacteria sporulate with 30% efficiency. Overexpression of the complete SpoIIE protein in a divIC mutant leads to significant σF activity, indicating that the septum requirement for activating σF can be bypassed. In contradiction to current models, we propose that genetic asymmetry is not created by unequal distribution of SpoIIE within the sporangium, but by exclusion of an inhibitor of SpoIIE from the forespore. This putative inhibitor would be a cytoplasmic molecule that interacts with SpoIIE and shuts off its phosphatase activity until it disappears specifically from the forespore.

Christensen, Henriette; Garton, Natalie J.; Horobin, Richard W.; Minnikin, David E.; Barer, Michael R.

doi: 10.1046/j.1365-2958.1999.01304.xpmid: 10200973

The complex mycobacterial cell envelope is recognized as a critical factor in our failure to control tuberculosis, leprosy and other non‐tuberculous pathogens. Although its composition has been extensively determined, many details regarding the organization of the envelope remain uncertain. This is particularly so for the non‐covalently bound lipids, whose natural distribution may be disrupted by conventional biochemical or cytological techniques. In order to study the native organization of lipid domains in the mycobacterial envelope, we have applied a range of fluorescent lipophilic probes to live mycobacteria, including Mycobacterium smegmatis, Mycobacterium tuberculosis, Mycobacterium avium, Mycobacterium gadium and Mycobacterium aurum, and analysed the resultant signals by fluorescence microscopy and digital image processing. Five key features were observed: (i) the presence of both envelope and intracellular lipid domains; (ii) differential localization of probes into these domains influenced predominantly by their hydrophobicity, as modelled by their calculated octanol:water partition coefficients and by their amphiphilicities; (iii) uneven distribution of lipophilic material in the envelope; (iv) selective labelling of septal regions of the envelope; and (v) modification of labelling patterns by additional treatments such as fluorescence quenching antibodies, detergents and solvents. Using this last approach, a coherent cell envelope lipid domain was demonstrated outside the cytoplasmic membrane and, for the first time, the proposed covalently linked mycolyl‐arabinogalactan‐peptidoglycan macromolecular complex was imaged directly. The use of fluorescent probes and high‐resolution fluorescence microscopy has enabled us to obtain a coherent view of distinct lipid domains in mycobacteria. Further application of this approach will facilitate understanding of the role of lipids in the physiology of these organisms.

Skurnik, Mikael; Venho, Reija; Bengoechea, José‐Antonio; Moriyón, Ignacio

doi: 10.1046/j.1365-2958.1999.01285.xpmid: 10200964

Lipopolysaccharide (LPS) of Yersinia enterocolitica O:3 has an inner core linked to both the O‐antigen and to an outer core hexasaccharide that forms a branch. The biological role of the outer core was studied using polar and non‐polar mutants of the outer core biosynthetic operon. Analysis of O‐antigen‐ and outer core‐deficient strains suggested a critical role for the outer core in outer membrane properties relevant in resistance to antimicrobial peptides and permeability to hydrophobic agents, and indirectly relevant in resistance to killing by normal serum. Wild‐type bacteria but not outer core mutants killed intragastrically infected mice, and the intravenous lethal dose was ≈104‐fold higher for outer core mutants. After intragastric infection, outer core mutants colonized Peyer's patches and invaded mesenteric lymph nodes, spleen and liver, and induced protective immunity against wild‐type bacteria. In mice co‐infected intragastrically with an outer core mutant–wild type mixture, both strains colonized Peyer's patches similarly during the first 2 days, but the mutant was much less efficient in colonizing deeper organs and was cleared faster from Peyer's patches. The results demonstrate that outer core is required for Y. enterocolitica O:3 full virulence, and strongly suggest that it provides resistance against defence mechanisms (most probably those involving bactericidal peptides).

Corral‐Debrinski, M.; Belgareh, N.; Blugeon, C.; Claros, M. G.; Doye, V.; Jacq, C.

doi: 10.1046/j.1365-2958.1999.01295.xpmid: 10200968

During evolution, cellular processes leading to the transfer of genetic information failed to send all the mitochondrial genes into the nuclear genome. Two mitochondrial genes are still exclusively located in the mitochondrial genome of all living organisms. They code for two highly hydrophobic proteins: the apocytochrome b and the subunit I of cytochrome oxidase. Assuming that the translocation machinery could not efficiently transport long hydrophobic fragments, we searched for multicopy suppressors of this physical blockage. We demonstrated that overexpression of Pse1p/Kap121p or Kap123p, which belong to the superfamily of karyopherin β proteins, facilitates the translocation of chimeric proteins containing several stretches of apocytochrome b fused to a reporter mitochondrial gene. The effect of PSE1/KAP121 overexpression (in which PSE1 is protein secretion enhancer 1) on mitochondrial import of the chimera is correlated with an enrichment of the corresponding transcript in cytoplasmic ribosomes associated with mitochondria. PSE1/KAP121 overexpression also improves the import of the hydrophobic protein Atm1p, an ABC transporter of the mitochondrial inner membrane. These results suggest that in vivo PSE1/KAP121 overexpression facilitates, either directly or indirectly, the co‐translational import of hydrophobic proteins into mitochondria.

Deschamps, Francine; Langin, Thierry; Maurer, Patricia; Gerlinger, Catherine; Felenbok, Béatrice; Daboussi, Marie‐Josée

doi: 10.1046/j.1365-2958.1999.01278.xpmid: 10200958

The Fot1 transposon is active in some strains of the plant pathogenic fungus Fusarium oxysporum. In a high‐copy‐number strain that contains autonomous elements, we have detected a transcript of 1.7 kb hybridizing to Fot1 in very low amounts. Mapping the 3′ and 5′ termini of this transcript confirms that it corresponds to a Fot1‐specific transcript. In this strain, five independent mutants of the transgene (niaD ) encoding nitrate reductase have arisen by insertion of Fot1 into the third intron. The analysis of the effect of Fot1 insertion in these mutants shows that, depending on the orientation of Fot1 relative to niaD, different truncated chimeric niaD–Fot1 transcripts are produced. Mapping the 5′ and 3′ ends of these transcripts reveals (i) premature polyadenylation at sites present in the 5′ and 3′ untranslated regions of Fot1, and (ii) initiation of some transcripts in the 3′ part of the niaD gene at sites located immediately downstream of the Fot1 insertion. Thus, a novel promoter, associated with the end of Fot1, directs transcriptional activity outwards from the element into the coding sequence of the niaD gene. These effects demonstrate that Fot1 insertion provides an additional general mechanism controlling fungal gene expression.

Wolfgang, Matthew; Van Putten, Jos P. M.; Hayes, Stanley F.; Koomey, Michael

doi: 10.1046/j.1365-2958.1999.01269.xpmid: 10200956

The expression of type IV pili (Tfp) by Neisseria gonorrhoeae has been shown to be essential for natural genetic transformation at the level of sequence‐specific uptake of DNA. All previously characterized mutants defective in this step of transformation either lack Tfp or are altered in the expression of Tfp‐associated properties, such as twitching motility, autoagglutination and the ability to bind to human epithelial cells. To examine the basis for this relationship, we identified potential genes encoding polypeptides sharing structural similarities to PilE, the Tfp subunit, within the N. gonorrhoeae genome sequence database. We found that disruption of one such gene, designated comP (for competence‐associated prepilin), leads to a severe defect in the capacity to take up DNA in a sequence‐specific manner, but does not alter Tfp biogenesis or expression of the Tfp‐associated properties of autoagglutination, twitching motility and human epithelial cell adherence. Indirect evidence based on immunodetection suggests that ComP is expressed at very low levels relative to that of PilE. The process of DNA uptake in gonococci, therefore, is now known to require the expression of at least three distinct components: Tfp, the recently identified PilT protein and ComP.

Odenbreit, Stefan; Till, Markus; Hofreuter, Dirk; Faller, Gerhard; Haas, Rainer

doi: 10.1046/j.1365-2958.1999.01300.xpmid: 10200971

In this study, we isolated and characterized a chromosomal locus of Helicobacter pylori previously identified by transposon shuttle mutagenesis as being involved in the adhesion of the pathogen to gastric epithelial cells. Two closely homologous genes were identified, designated as alpA and alpB, encoding outer membrane (OM) proteins of 518 amino acids each. They are members of the outer membrane protein supergene family identified in the H. pylori 26695 complete genome sequence. AlpA carries a functional lipoprotein signal sequence. AlpB carries a putative standard N‐terminal signal sequence and shows a strong amino‐acid sequence identity to AlpA. Transposon insertion mutagenesis, immunoblotting and primer extension studies indicate that both genes are organized in an operon, but no obvious consensus promoter sequence was found upstream of the transcriptional start site. The C‐terminal portion of both proteins is predicted to form a porin‐like β‐barrel in the outer membrane, consisting of 14 transmembrane amphipathic β‐strands. Adhesion experiments with defined isogenic mutants indicate that both proteins are necessary for specific adherence of H. pylori to human gastric tissue. The pattern of AlpAB‐dependent adherence of H. pylori to the gastric epithelial surface shows a clear difference to the BabA2‐mediated adherence to Lewisb, suggesting that a different receptor is involved.

Bae, Weonhye; Phadtare, Sangita; Severinov, Konstantin; Inouye, Masayori

doi: 10.1046/j.1365-2958.1999.01284.xpmid: 10200963

Escherichia coli contains nine members of the CspA protein family from CspA to CspI. To elucidate the cellular function of CspE, we constructed a ΔcspE strain. CspE is highly produced at 37°C. The synthesis level of CspE transiently increased during the growth lag period after dilution of stationary‐phase cells into the fresh medium at 37°C. This is consistent with the ΔcspE phenotype of the longer growth lag period after dilution. The protein synthesis patterns of the ΔcspE strain and the wild‐type strain were compared using two‐dimensional gel electrophoresis. In the ΔcspE strain, the synthesis of a number of proteins at 37°C was found to be altered and cspA was derepressed. The derepression of cspA in the ΔcspE strain was at the level of transcription in a promoter‐independent fashion but was not caused by stabilization of the cspA mRNA, which was shown to be a major cause of CspA induction after cold shock. In vitro transcription assays demonstrated that both CspE and CspA enhanced transcription pause at the region immediately downstream of the cold box, a putative repressor binding site on the cspA mRNA. In a cell‐free protein synthesis system using S‐30 cell extracts, CspA production was specifically inhibited by the addition of CspE. These results indicate that CspE functions as a negative regulator for cspA expression at 37°C, probably by interacting with the transcription elongation complex at the cspA cold box region.

Zhang, Jing‐Ren; Idanpaan‐Heikkila, Ilona; Fischer, Werner; Tuomanen, Elaine I.

doi: 10.1046/j.1365-2958.1999.01291.xpmid: 10200966

Phosphorylcholine is an important bioactive adduct to the teichoic acid (TA) and lipoteichoic acid (LTA) of the surface of Streptococcus pneumoniae. We have identified and characterized a genetic locus lic that is required for phosphorylcholine metabolism in S. pneumoniae. The pneumococcal lic locus consists of eight genes, licA, licB, licC and licD1, licD2 and three additional open reading frames. Pneumococcal licA, licB, licC, licD1 and licD2 have significant sequence similarity to licA, licB, licC and licD of Haemophilus influenzae. Mutation of licD2 led to decreased (3H)‐choline uptake, aberrant migration of LTA chains in SDS–PAGE gels, loss of several surface proteins, and a phase‐locked hypertransparent colony phenotype. Moreover, the licD2− mutant failed to undergo lysis after treatment with penicillin at high cell density and showed decreased transformation competence. Finally, the licD2− mutant demonstrated decreased adherence to the human type II alveolar cells, reduced nasopharyngeal colonization in infant rats, as well as significantly impaired virulence upon intraperitoneal challenge of CF1 mice. Identification of the lic genes in the pneumococcus will facilitate further characterization of the role of surface choline in microbial physiology and pathogenesis.