Legionella pneumophila requires polyamines for optimal intracellular growth

The Gram-negative intracellular pathogen Legionella pneumophila replicates in a membrane-bound compartment known as the Legionella-containing vacuole (LCV), into which it abundantly releases its chaperonin, HtpB. To determine whether HtpB remains within the LCV or reaches the host cell cytoplasm, we infected U937 human macrophages and CHO cells with L. pneumophila expressing a translocation reporter consisting of the Bordetella pertussisa denylate cyclase fused to HtpB. These infections led to increased cyclic AMP levels, suggesting that HtpB reaches the host cell cytoplasm. To identify potential functions of cytoplasmic HtpB, we expressed it in the yeast Saccharomyces cerevisiae, where HtpB induced pseudohyphal growth. A yeast-two-hybrid screen showed that HtpB interacted with S-adenosylmethionine decarboxylase (SAMDC), an essential yeast enzyme (encoded by SPE2) that is required for polyamine biosynthesis. Increasing the copy number of SPE2 induced pseudohyphal growth in S. cerevisiae; thus, we speculated that (i) HtpB induces pseudohyphal growth by activating polyamine synthesis and (ii) L. pneumophila may require exogenous polyamines for growth. A pharmacological inhibitor of SAMDC significantly reduced L. pneumophila replication in L929 mouse cells and U937 macrophages, whereas exogenously added polyamines moderately favored intracellular growth, confirming that polyamines and host SAMDC activity promote L. pneumophila proliferation. Bioinformatic analysis revealed that most known enzymes required for polyamine biosynthesis in bacteria (including SAMDC) are absent in L. pneumophila, further suggesting a need for exogenous polyamines. We hypothesize that HtpB may function to ensure a supply of polyamines in host cells, which are required for the optimal intracellular growth of L. pneumophila.     

Prevalence of the multiple antibiotic resistance operon (marRAB) in the genus Salmonella

The multiple antibiotic resistance operon (marRAB) is a member of the multidrug resistance (mdr) systems. Similar to other mdr systems, this operon when induced encodes resistance to structurally and functionally unrelated antibiotics. marRAB has been shown to be conserved in the family Enterobacteriaceae, but within the genus Salmonella certain species appeared to be lacking this operon. To investigate how conserved the marRAB operon was in Salmonella, 30 veterinary isolates were examined by PCR, Southern blot, and dot blot analysis. Using DNA primers based on the marRAB operon of S. typhimurium, a predicted 2.3-kb amplicon resulted after PCR in 16 of the 30 organisms. The 2.3-kb DNA band from S. enteritidis was cloned and sequenced and shown to possess 99% sequence homology to marRAB from S. typhimurium. Using a labeled marRAB gene probe from S. enteritidis, Southern blot and dot blot analysis confirmed the presence of the operon in all 30 Salmonella species examined. Furthermore, when these isolates were induced with low levels of either tetracycline or chloramphenicol, increased antimicrobial resistance was observed to structurally and functionally unrelated antibiotics. Thus, the widespread occurrence of the marRAB locus in this genus prescribes judicious use of antimicrobials to avoid induction of a mdr phenotype.     

Overexpression of the Bt cry2Aa2 operon in chloroplasts leads to formation of insecticidal crystals

In nuclear transgenic plants, expression of multiple genes requires introduction of individual genes and time-consuming subsequent backcrosses to reconstitute multi-subunit proteins or pathways, a problem that is compounded by variable expression levels. In order to accomplish expression of multiple genes in a single transformation event, we have introduced several genes into the chromoplast genome. We confirmed stable integration of the cry2Aa2 operon by PCR and Southern blot analyses in T(0) and T(1) transgenic plants. Foreign protein accumulated at 45.3% of the total soluble protein in mature leaves and remained stable even in old bleached leaves (46.1%), thereby increasing the efficacy and safety of transgenic plants throughout the growing season. This represents the highest level of foreign gene expression reported in transgenic plants to date. Insects that are normally difficult to control (10-day old cotton bollworm, beet armyworm) were killed 100% after consuming transgenic leaves. Electron micrographs showed the presence of the insecticidal protein folded into cuboidal crystals. Formation of crystals of foreign proteins (due to hyperexpression and folding by the putative chaperonin, ORF 2) provides a simple method of purification by centrifugation and enhances stability by protection from cellular proteases. Demonstration of expression of an operon in transgenic plants paves the way to engineering new pathways in plants in a single transformation event.     

Identification of Elements That Dictate the Specificity of Mitochondrial Hsp60 for Its Co-Chaperonin

Type I chaperonins (cpn60/Hsp60) are essential proteins that mediate the folding of proteins in bacteria, chloroplast and mitochondria. Despite the high sequence homology among chaperonins, the mitochondrial chaperonin system has developed unique properties that distinguish it from the widely-studied bacterial system (GroEL and GroES). The most relevant difference to this study is that mitochondrial chaperonins are able to refold denatured proteins only with the assistance of the mitochondrial co-chaperonin. This is in contrast to the bacterial chaperonin, which is able to function with the help of co-chaperonin from any source. The goal of our work was to determine structural elements that govern the specificity between chaperonin and co-chaperonin pairs using mitochondrial Hsp60 as model system. We used a mutagenesis approach to obtain human mitochondrial Hsp60 mutants that are able to function with the bacterial co-chaperonin, GroES. We isolated two mutants, a single mutant (E321K) and a double mutant (R264K/E358K) that, together with GroES, were able to rescue an E. coli strain, in which the endogenous chaperonin system was silenced. Although the mutations are located in the apical domain of the chaperonin, where the interaction with co-chaperonin takes place, none of the residues are located in positions that are directly responsible for co-chaperonin binding. Moreover, while both mutants were able to function with GroES, they showed distinct functional and structural properties. Our results indicate that the phenotype of the E321K mutant is caused mainly by a profound increase in the binding affinity to all co-chaperonins, while the phenotype of R264K/E358K is caused by a slight increase in affinity toward co-chaperonins that is accompanied by an alteration in the allosteric signal transmitted upon nucleotide binding. The latter changes lead to a great increase in affinity for GroES, with only a minor increase in affinity toward the mammalian mitochondrial co-chaperonin.     

Anthrose biosynthetic operon of Bacillus anthracis

The exosporium of Bacillus anthracis spores consists of a basal layer and an external hair-like nap. The nap is composed primarily of the glycoprotein BclA, which contains a collagen-like region with multiple copies of a pentasaccharide side chain. This oligosaccharide possesses an unusual terminal sugar called anthrose, followed by three rhamnose residues and a protein-bound N-acetylgalactosamine. Based on the structure of anthrose, we proposed an enzymatic pathway for its biosynthesis. Examination of the B. anthracis genome revealed six contiguous genes that could encode the predicted anthrose biosynthetic enzymes. These genes are transcribed in the same direction and appear to form two operons. We introduced mutations into the B. anthracis chromosome that either delete the promoter of the putative upstream, four-gene operon or delete selected genes in both putative operons. Spores produced by strains carrying mutations in the upstream operon completely lacked or contained much less anthrose, indicating that this operon is required for anthrose biosynthesis. In contrast, inactivation of the downstream, two-gene operon did not alter anthrose content. Additional experiments confirmed the organization of the anthrose operon and indicated that it is transcribed from a sigma(E)-specific promoter. Finally, we demonstrated that anthrose biosynthesis is not restricted to B. anthracis as previously suggested.     

The Escherichia coli groE chaperonins

The E.coli groES and groEL genes have been shown to form an operon, to be essential for E. coli viability, and to belong to the so-called heat-shock class of genes whose expression is regulated by the intracellular levels of sigma factor sigma 32. Both groE chaperonin proteins possess a seven-fold axis of symmetry, groES being composed of seven identical subunits of 97 amino acids each, and groEL of fourteen identical subunits of 548 amino acids each. The two groE chaperonins interact intimately as judged by both genetic and biochemical criteria. This interaction has been shown to be required for both bacteriophage morphogenesis and bacterial growth. The groEL chaperonin has been shown to bind to a number of incomplete or unfolded polypeptides in vitro. Such binding may prevent misfolding and promote rapid intra- or intermolecular folding of polypeptides in vivo. The proposed role of the groES chaperonin is to displace the polypeptides bound to groEL, thus effectively promoting the recycling of groEL.     

gltBDF operon of Escherichia coli.

A 2.0-kilobase DNA fragment carrying antibiotic resistance markers was inserted into the gltB gene of Escherichia coli previously cloned in a multicopy plasmid. Replacement of the chromosomal gltB+ gene by the gltB225::omega mutation led to cells unable to synthesize glutamate synthase, utilize growth rate-limiting nitrogen sources, or derepress their glutamine synthetase. The existence of a gltBDF operon encoding the large (gltB) and small (gltD) subunits of glutamate synthase and a regulatory peptide (gltF) at 69 min of the E. coli linkage map was deduced from complementation analysis. A plasmid carrying the entire gltB+D+F+ operon complemented cells for all three of the mutant phenotypes associated with the polar gltB225::omega mutation in the chromosome. By contrast, plasmids carrying gltB+ only complemented cells for glutamate synthase activity. A major tricistronic mRNA molecule was detected from Northern (RNA blot) DNA-RNA hybridization experiments with DNA probes containing single genes of the operon. A 30,200-dalton polypeptide was identified as the gltF product, the lack of which was responsible for the inability of cells to use nitrogen-limiting sources associated with gltB225::omega.     

The DotL protein, a member of the TraG-coupling protein family, is essential for Viability of Legionella pneumophila strain Lp02

Legionella pneumophila is able to survive inside phagocytic cells by an internalization route that bypasses fusion of the nascent phagosome with the endocytic pathway to allow formation of a replicative phagosome. The dot/icm genes, a major virulence system of L. pneumophila, encode a type IVB secretion system that is required for intracellular growth. One Dot protein, DotL, has sequence similarity to type IV secretion system coupling proteins (T4CPs). In other systems, coupling proteins are not required for viability of the organism. Here we report the first example of a strain, L. pneumophila Lp02, in which a putative T4CP is essential for viability of the organism on bacteriological media. This result is particularly surprising since the majority of the dot/icm genes in Lp02 are dispensable for growth outside of a host cell, a condition that does not require a functional Dot/Icm secretion complex. We were able to isolate suppressors of the Delta dotL lethality and found that many contained mutations in other components of the Dot/Icm secretion system. A systematic analysis of dot/icm deletion mutants revealed that the majority of them (20 of 26) suppressed the lethality phenotype, indicating a partially assembled secretion system may be the source of Delta dotL toxicity in the wild-type strain. These results are consistent with a model in which the DotL protein plays a role in regulating the activity of the L. pneumophila type IV secretion apparatus.     

A mobile loop order-disorder transition modulates the speed of chaperonin cycling

Molecular machines order and disorder polypeptides as they form and dissolve large intermolecular interfaces, but the biological significance of coupled ordering and binding has been established in few, if any, macromolecular systems. The ordering and binding of GroES co-chaperonin mobile loops accompany an ATP-dependent conformational change in the GroEL chaperonin that promotes client protein folding. Following ATP hydrolysis, disordering of the mobile loops accompanies co-chaperonin dissociation, reversal of the GroEL conformational change, and release of the client protein. "High-affinity" GroEL mutants were identified by their compatibility with "low-affinity" co-chaperonin mutants and incompatibility with high-affinity co-chaperonin mutants. Analysis of binding kinetics using the intrinsic fluorescence of tryptophan-containing co-chaperonin variants revealed that excessive affinity causes the chaperonin to stall in a conformation that forms in the presence of ATP. Destabilizing the beta-hairpins formed by the mobile loops restores the normal rate of dissociation. Thus, the free energy of mobile-loop ordering and disordering acts like the inertia of an engine's flywheel by modulating the speed of chaperonin conformational changes.     

Evaluation of the rrn operon copy number in Bifidobacterium using real-time PCR

AIMS: A real-time PCR-based method was developed to evaluate the Bifidobacterium rRNA operon copy number. As a result of their repetitive nature, rRNA operons are very suitable targets for chromosomal integration of heterologous genes. METHODS AND RESULTS: The rrn operon multiplicity per chromosome was determined by real-time PCR quantification of the 16S rRNA amplicons obtained from genomic DNA. The values obtained in several bifidobacterial strains of human origin ranged from 1 to 5. The reliability of the method developed was confirmed by Southern hybridization technique. CONCLUSIONS: In the Bifidobacterium genus the rrn operon copies showed variability at species and strain level. The identification of Bifidobacterium strains with high rRNA multiplicity allowed the selection of potential hosts for chromosomal integration. SIGNIFICANCE AND IMPACT OF THE STUDY: The methodology here proposed represents a rapid, reliable and sensitive new tool for the quantification of rrn operon copy number in bacteria.     

Regulation of hypercompetence in Legionella pneumophila

Although many bacteria are known to be naturally competent for DNA uptake, this ability varies dramatically between species and even within a single species, some isolates display high levels of competence while others seem to be completely nontransformable. Surprisingly, many nontransformable bacterial strains appear to encode components necessary for DNA uptake. We believe that many such strains are actually competent but that this ability has been overlooked because standard laboratory conditions are inappropriate for competence induction. For example, most strains of the gram-negative bacterium Legionella pneumophila are not competent under normal laboratory conditions of aerobic growth at 37 degrees C. However, it was previously reported that microaerophilic growth at 37 degrees C allows L. pneumophila serogroup 1 strain AA100 to be naturally transformed. Here we report that another L. pneumophila serogroup 1 strain, Lp02, can also be transformed under these conditions. Moreover, Lp02 can be induced to high levels of competence by a second set of conditions, aerobic growth at 30 degrees C. In contrast to Lp02, AA100 is only minimally transformable at 30 degrees C, indicating that Lp02 is hypercompetent under these conditions. To identify potential causes of hypercompetence, we isolated mutants of AA100 that exhibited enhanced DNA uptake. Characterization of these mutants revealed two genes, proQ and comR, that are involved in regulating competence in L. pneumophila. This approach, involving the isolation of hypercompetent mutants, shows great promise as a method for identifying natural transformation in bacterial species previously thought to be nontransformable.