- Volume 157, Issue 10, 2011

Rhizobia are a disparate collection of soil bacteria capable of reducing atmospheric nitrogen in symbiosis with legumes (Fix phenotype). Synthesis of the nitrogenase and its accessory components is under the transcriptional control of the key regulator NifA and is generally restricted to the endosymbiotic forms of rhizobia known as bacteroids. Amongst studied rhizobia, Sinorhizobium fredii strain NGR234 has the remarkable ability to fix nitrogen in association with more than 130 species in 73 legume genera that form either determinate, indeterminate or aeschynomenoid nodules. Hence, NGR234 is a model organism to study nitrogen fixation in association with a variety of legumes. The symbiotic plasmid pSfrNGR234a carries more than 50 genes that are under the transcriptional control of NifA. To facilitate the functional analysis of NifA-regulated genes a new transposable element, TnEKm-PwA, was constructed. This transposon combines the advantages of in vitro mutagenesis of cloned DNA fragments with a conditional read-out promoter from NGR234 (PwA) that reinitiates NifA-dependent transcription downstream of transposition sites. To test the characteristics of the new transposon, the nifQdctA1y4vGHIJ operon was mutated using either the Omega interposon or TnEKm-PwA. The symbiotic phenotypes on various hosts as well as the transcriptional characteristics of these mutants were analysed in detail and compared with the ineffective (Fix−) phenotype of strain NGRΔnifA, which lacks a functional copy of nifA. De novo transcription from inserted copies of TnEKm-PwA inside bacteroids was confirmed by qRT-PCR. Unexpectedly, polar mutants in dctA1 and nifQ were Fix+ on all of the hosts tested, indicating that none of the six genes of the nifQ operon of NGR234 is essential for symbiotic nitrogen fixation on plants that form nodules of either determinate or indeterminate types.

Chlamydia trachomatis is the most common bacterial infection of the human reproductive tract globally; however, the mechanisms underlying the adaptation of the organism to its natural target cells, human endocervical epithelial cells, are not clearly understood. To secure its intracellular niche, C. trachomatis must modulate the host cellular machinery by secreting virulence factors into the host cytosol to facilitate bacterial growth and survival. Here we used primary human endocervical epithelial cells and HeLa cells infected with C. trachomatis to examine the secretion of bacterial proteins during productive growth and persistent growth induced by ampicillin. Specifically, we observed a decrease in secretable chlamydial protease-like activity factor (CPAF) in the cytosol of host epithelial cells exposed to ampicillin with no evident reduction of CPAF product by C. trachomatis. In contrast, the expression of CopN and Tarp was downregulated, suggesting that C. trachomatis responds to ampicillin exposure by selectively altering the expression of secretable proteins. In addition, we observed a greater accumulation of outer-membrane vesicles from C. trachomatis in persistently infected cells. Taken together, these results suggest that the regulation of both gene expression and the secretion of chlamydial virulence proteins is involved in the adaptation of the bacteria to a persistent infection state in human genital epithelial cells.

By the analysis of the Aeromonas hydrophila ATCC7966T genome we identified A. hydrophila AH-3 MotY. A. hydrophila MotY, like MotX, is essential for the polar flagellum function energized by an electrochemical potential of Na+ as coupling ion, but is not involved in lateral flagella function energized by the proton motive force. Thus, the A. hydrophila polar flagellum stator is a complex integrated by two essential proteins, MotX and MotY, which interact with one of two redundant pairs of proteins, PomAB and PomA2B2. In an A. hydrophila motX mutant, polar flagellum motility is restored by motX complementation, but the ability of the A. hydrophila motY mutant to swim is not restored by introduction of the wild-type motY alone. However, its polar flagellum motility is restored when motX and -Y are expressed together from the same plasmid promoter. Finally, even though both the redundant A. hydrophila polar flagellum stators, PomAB and PomA2B2, are energized by the Na+ ion, they cannot be exchanged. Furthermore, Vibrio parahaemolyticus PomAB and Pseudomonas aeruginosa MotAB or MotCD are unable to restore swimming motility in A. hydrophila polar flagellum stator mutants.

The Plasmodium falciparum kinome includes a family of four protein kinases (Pfnek-1 to -4) related to the NIMA (never-in-mitosis) family, members of which play important roles in mitosis and meiosis in eukaryotic cells. Only one of these, Pfnek-1, which we previously characterized at the biochemical level, is expressed in asexual parasites. The other three (Pfnek-2, -3 and -4) are expressed predominantly in gametocytes, and a role for nek-2 and nek-4 in meiosis has been documented. Here we show by reverse genetics that Pfnek-1 is required for completion of the asexual cycle in red blood cells and that its expression in gametocytes in detectable by immunofluorescence in male (but not in female) gametocytes, in contrast with Pfnek-2 and Pfnek-4. This indicates that the function of Pfnek-1 is non-redundant with those of the other members of the Pfnek family and identifies Pfnek-1 as a potential target for antimalarial chemotherapy. A medium-throughput screen of a small-molecule library provides proof of concept that recombinant Pfnek-1 can be used as a target in drug discovery.

A large number of polypeptides are attached to poly(3-hydroxybutyrate) (PHB) granules of Ralstonia eutropha, such as PHB synthase (PhaC1), several PHB depolymerases (PhaZs) and phasins (PhaPs), the regulator protein PhaR Reu , and possibly others. In this study we used the bacterial adenylate cyclase-based two-hybrid assay to investigate interactions between known PHB granule-associated proteins (PGAPs) and to screen for new PGAPs. The utility of the system was tested by the in vivo verification of previously postulated interactions of the PHB synthase subunits of R. eutropha (PhaC1 homo-oligomerization) and of Bacillus megaterium (PhaC Bmeg –PhaR Bmeg hetero-oligomerization). Nine proteins (PhaA, PhaB1, PhaC1, PhaP1–PhaP4, PhaZ1 and PhaR), with established functions in PHB metabolism of R. eutropha, were tested for interaction in all combinations. While no significant interaction was detected between the PHB synthase PhaC1 and any of the other eight tested Pha proteins, strong interactions were found between all phasin proteins, in particular between PhaP2 and PhaP4. When PhaP2 was used as bait in a two-hybrid screening experiment with a genomic library of R. eutropha, the B1934 gene product was identified in 24 out of 53 isolated clones. B1934 encodes a hypothetical protein (15.7 kDa) with similarity to phasins of PHB-accumulating bacteria. A fusion protein of eYfp and the B1934 gene product colocalized with PHB granules, confirming that B1934 represents a new phasin (PhaP5). PhaP5 was not essential for PHB granule formation, but overexpression of PhaP5 increased the number of cells with PHB granules at the cell poles.

In trace amounts, copper is essential for the function of key enzymes in prokaryotes and eukaryotes. Organisms have developed sophisticated mechanisms to control the cytosolic level of the metal, manage its toxicity and survive in copper-rich environments. Here we show that the Sulfolobus CopR represents a novel class of copper-responsive regulators, unique to the archaeal domain. Furthermore, by disruption of the ORF Sso2652 (copR) of the Sulfolobus solfataricus genome, we demonstrate that the gene encodes a transcriptional activator of the copper-transporting ATPase CopA gene and co-transcribed copT, encoding a putative copper-binding protein. Disruption resulted in a loss of copper tolerance in two copR-knockout mutants, while metals such as zinc, cadmium and chromium did not affect their growth. Copper sensitivity in the mutant was linked to insufficient levels of expression of CopA and CopT. The findings were further supported by time-course inductively coupled plasma optical emission spectrometry measurements, whereby continued accumulation of copper in the S. solfataricus mutant was observed. In contrast, copper accumulation in the wild-type stabilized after reaching approximately 6 pg (µg total protein)–1. Complementation of the disrupted mutant with a wild-type copy of the copR gene restored the wild-type phenotype with respect to the physiological and transcriptional response to copper. These observations, taken together, lead us to propose that CopR is an activator of copT and copA transcription, and the member of a novel class of copper-responsive regulators.

Crithidia deanei is a trypanosomatid protozoan that harbours a symbiotic bacterium. The partners maintain a mutualistic relationship, thus constituting an excellent model for studying metabolic exchanges between the host and the symbiont, the origin of organelles and cellular evolution. According to molecular analysis, symbionts of different trypanosomatid species share high identity and descend from a common ancestor, a β-proteobacterium of the genus Bordetella. The endosymbiont is surrounded by two membranes, like Gram-negative bacteria, but its envelope presents special features, since phosphatidylcholine is a major membrane component and the peptidoglycan layer is highly reduced, as described in other obligate intracellular bacteria. Like the process that generated mitochondria and plastids, the endosymbiosis in trypanosomatids depends on pathways that facilitate the intensive metabolic exchanges between the bacterium and the host protozoan. A search of the annotated symbiont genome database identified one sequence with identity to porin-encoding genes of the genus Bordetella. Considering that the symbiont outer membrane has a great accessibility to cytoplasm host factors, it was important to characterize this single porin-like protein using biochemical, molecular, computational and ultrastructural approaches. Antiserum against the recombinant porin-like molecule revealed that it is mainly located in the symbiont envelope. Secondary structure analysis and comparative modelling predicted the protein 3D structure as an 18-domain β-barrel, which is consistent with porin channels. Electrophysiological measurements showed that the porin displays a slight preference for cations over anions. Taken together, the data presented herein suggest that the C. deanei endosymbiont porin is phylogenetically and structurally similar to those described in Gram-negative bacteria, representing a diffusion channel that might contribute to the exchange of nutrients and metabolic precursors between the symbiont and its host cell.

The conversion of nicotinamide to nicotinic acid by nicotinamidase enzymes is a critical step in maintaining NAD+ homeostasis and contributes to numerous important biological processes in diverse organisms. In Borrelia burgdorferi, the nicotinamidase enzyme, PncA, is required for spirochaete survival throughout the infectious cycle. Mammals lack nicotinamidases and therefore PncA may serve as a therapeutic target for Lyme disease. Contrary to the in vivo importance of PncA, the current annotation for the pncA ORF suggests that the encoded protein may be inactive due to the absence of an N-terminal aspartic acid residue that is a conserved member of the catalytic triad of characterized PncA proteins. Herein, we have used genetic and biochemical strategies to determine the N-terminal sequence of B. burgdorferi PncA. Our data demonstrate that the PncA protein is 24 aa longer than the currently annotated sequence and that pncA translation is initiated from the rare, non-canonical initiation codon AUU. These findings are an important first step in understanding the catalytic function of this in vivo-essential protein.

The integrase IntI1 catalyses recombination of antibiotic-resistance gene cassettes in the integron, a widely found bacterial mobile element active in spreading antibiotic multi-resistance. We have previously shown that resistance cassette recombination rate and specificity depend on the amount of intracellular integrase. Here, we used in vivo and in vitro methods to examine convergent expression of the integrase promoter (Pint ) and of the cassette promoters (Pc and P2 ) in the prototypical plasmid-borne class 1 integron, In2. Highly conserved Pint has near consensus −10 and −35 hexamers for σ70 RNA polymerase, but there are 11 naturally occurring arrangements of Pc alone or combinations of the Pc +P2 cassette promoters (note that P2 occurs with a 14 or 17 bp spacer). Using a bi-directional reporter vector, we found that Pint is a strong promoter in vivo, but its expression is reduced by converging transcription from Pc and P2 . In addition to cis-acting convergence control of integrase expression, the regulator site prediction program, prodoric 8.9, identified sites for global regulators FIS, LexA, IHF and H-NS in and near the integron promoters. In strains mutated in each global regulator, we found that: (1) FIS repressed integrase and cassette expression; (2) LexA repressed Pint and P2 with the 14 bp spacer version of P2 and FIS was necessary for maximum LexA repression; (3) IHF activated Pint when it faced the strong 17 bp spacer P2 but did not elevate its expression versus LexA-repressed P2 with the 14 bp spacer; and (4) H-NS repressed both Pint and the 14 bp P2 but activated the 17 bp P2 cassette promoters. Mobility shift assays showed that FIS and IHF interact directly with the promoter regions and DNase I footprinting confirmed extensive protection by FIS of wild-type In2 integron promoter sequence. Thus, nucleoid-associated proteins, known to act directly in site-specific recombination, also control integron gene expression directly and possibly indirectly.

The human pathogen Streptococcus pneumoniae harbours many genes encoding phosphotransferase systems and sugar ABC (ATP-binding cassette) transporters, including systems for the utilization of the β-glucoside sugar cellobiose. In this study, we show that the transcriptional regulator CelR, which has previously been found to be important for pneumococcal virulence, activates the expression of the cellobiose-utilization gene cluster (cel locus) of S. pneumoniae. Expression directed by the two promoters present in the cel locus was increased in the presence of cellobiose as sole carbon source in the medium, while expression decreased in the presence of glucose in the medium. Furthermore, we have predicted a 22 bp putative CelR regulatory site (5′-YTTTCCWTAWCAWTWAGGAAAA-3′) in the promoters of celA and celB, and in silico analysis showed that it is highly conserved in other pathogenic streptococci as well. Promoter truncations of celA and celB, where the half or full CelR regulatory site was deleted, confirmed that the CelR-binding site in PcelA and PcelB is functional. Transcriptome studies with the celR mutant and in silico prediction of the CelR regulatory site in the entire D39 genome sequence show that the cel locus is the only cluster of genes under the direct control of CelR. Therefore, CelR is a regulator dedicated to the cellobiose-dependent transcriptional activation of the cel locus.

The attachment organelles of bacterial species belonging to the Mycoplasma pneumoniae phylogenetic cluster are required for host cytadherence, gliding motility and virulence. Despite being closely related, these bacteria possess distinct cellular morphologies and gliding characteristics. The molecular mechanisms for most attachment organelle phenotypes, including shape and ability to power motility, are obscure. The attachment organelle-associated P30 protein of M. pneumoniae is implicated in both adherence and motility, with mutations negatively impacting cell morphology, adherence, gliding and virulence. To test whether the P30 alleles of different mycoplasma species confer species-specific attachment organelle properties, we created an M. pneumoniae strain in which the Mycoplasma genitalium P30 orthologue, P32, was substituted for the native P30. Selected clones were visualized by scanning electron microscopy to assess morphology and by indirect immunofluorescence microscopy to localize P32. Cytadherence ability and gliding motility were assessed by haemadsorption assay and phase-contrast microcinematography, respectively. Cell and attachment organelle morphologies were indistinguishable from wild-type M. pneumoniae as well as M. pneumoniae II-3 expressing a C-terminally 6×His-tagged P30 construct. P32 was localized to the tip of the attachment organelle of transformant cells. Although a specific role for P30 in species-specific phenotypes was not identified, this first test of orthologous gene replacement in different mycoplasma species demonstrates that the differences in the M. pneumoniae and M. genitalium proteins contribute little if anything to the different attachment organelle phenotypes between these species.

Transcription of rRNAs in Escherichia coli is directed from seven redundant rRNA operons, which are mainly regulated by their P1 promoters. Here we demonstrate by in vivo measurements that the amounts of individual rRNAs transcribed from the different operons under normal growth vary noticeably although the structures of all the P1 promoters are very similar. Moreover, we show that starvation for amino acids does not affect the seven P1 promoters in the same way. Notably, reduction of transcription from rrnD P1 was significantly lower compared to the other P1 promoters. The presence of DksA was shown to be crucial for the ppGpp-dependent downregulation of all P1 promoters. Because rrnD P1 is the only rrn promoter starting with GTP instead of ATP, we performed studies with a mutant rrnD promoter, where the initiating G+1 is replaced by A+1. These analyses demonstrated that the ppGpp sensitivity of rrn P1 promoters depends on the nature and concentration of initiating nucleoside triphosphates (iNTPs). Our results support the notion that the seven rRNA operons are differentially regulated and underline the importance of a concerted activity between ppGpp, DksA and an adequate concentration of the respective iNTP.

The ClpXP proteolytic complex is critical for maintaining cellular homeostasis, as well as expression of virulence properties. However, with the exception of the Spx global regulator, the molecular mechanisms by which the ClpXP complex exerts its influence in Streptococcus mutans are not well understood. Here, microarray analysis was used to provide novel insights into the scope of ClpXP proteolysis in S. mutans. In a ΔclpP strain, 288 genes showed significant changes in relative transcript amounts (P≤0.001, twofold cut-off) as compared with the parent. Similarly, 242 genes were differentially expressed by a ΔclpX strain, 113 (47 %) of which also appeared in the ΔclpP microarrays. Several genes associated with cell growth were downregulated in both mutants, consistent with the slow-growth phenotype of the Δclp strains. Among the upregulated genes were those encoding enzymes required for the biosynthesis of intracellular polysaccharides (glg genes) and malolactic fermentation (mle genes). Enhanced expression of glg and mle genes in ΔclpP and ΔclpX strains correlated with increased storage of intracellular polysaccharide and enhanced malolactic fermentation activity, respectively. Expression of several genes known or predicted to be involved in competence and mutacin production was downregulated in the Δclp strains. Follow-up transformation efficiency and deferred antagonism assays validated the microarray data by showing that competence and mutacin production were dramatically impaired in the Δclp strains. Collectively, our results reveal the broad scope of ClpXP regulation in S. mutans homeostasis and identify several virulence-related traits that are influenced by ClpXP proteolysis.

Here we tested the hypothesis that species of the soil-inhabiting insect-pathogenic fungus Metarhizium are not randomly distributed in soils but show plant-rhizosphere-specific associations. We isolated Metarhizium from plant roots at two sites in Ontario, Canada, sequenced the 5′ EF-1α gene to discern Metarhizium species, and developed an RFLP test for rapid species identification. Results indicated a non-random association of three Metarhizium species (Metarhizium robertsii, Metarhizium brunneum and Metarhizium guizhouense) with the rhizosphere of certain types of plant species (identified to species and categorized as grasses, wildflowers, shrubs and trees). M. robertsii was the only species that was found associated with grass roots, suggesting a possible exclusion of M. brunneum and M. guizhouense. Supporting this, in vitro experiments showed that M. robertsii conidia germinated significantly better in Panicum virgatum (switchgrass) root exudate than did M. brunneum or M. guizhouense. M. guizhouense and M. brunneum only associated with wildflower rhizosphere when co-occurring with M. robertsii. With the exception of these co-occurrences, M. guizhouense was found to associate exclusively with the rhizosphere of tree species, predominantly Acer saccharum (sugar maple), while M. brunneum was found to associate exclusively with the rhizosphere of shrubs and trees. These associations demonstrate that different species of Metarhizium associate with specific plant types.

Syntrophic growth involves the oxidation of organic compounds and subsequent transfer of electrons to an H2- or formate-consuming micro-organism. In order to identify genes involved specifically in syntrophic growth, a mutant library of Desulfovibrio alaskensis G20 was screened for loss of the ability to grow syntrophically with Methanospirillum hungatei JF-1. A collection of 20 mutants with an impaired ability to grow syntrophically was obtained. All 20 mutants grew in pure culture on lactate under sulfidogenic conditions at a rate and to a maximum OD600 similar to those of the parental strain. The largest number of mutations that affected syntrophic growth with lactate was in genes encoding proteins involved in H2 oxidation, electron transfer, hydrogenase post-translational modification, pyruvate degradation and signal transduction. The qrcB gene, encoding a quinone reductase complex (Qrc), and cycA, encoding the periplasmic tetrahaem cytochrome c 3 (TpIc3), were required by G20 to grow syntrophically with lactate. A mutant in the hydA gene, encoding an Fe-only hydrogenase (Hyd), is also impaired in syntrophic growth with lactate. The other mutants grew more slowly than the parental strain in syntrophic culture with M. hungatei JF-1. qrcB and cycA were shown previously to be required for growth of G20 pure cultures with H2 and sulfate. Washed cells of the parental strain produced H2 from either lactate or pyruvate, but washed cells of qrcB, cycA and hydA mutants produced H2 at rates similar to the parental strain from pyruvate and did not produce significant amounts of H2 from lactate. Real-time quantitative PCR assays showed increases in expression of the above three genes during syntrophic growth compared with pure-culture growth with lactate and sulfate. Our work shows that Hyd, Qrc and TpIc3 are involved in H2 production during syntrophic lactate metabolism by D. alaskensis G20 and emphasizes the importance of H2 production for syntrophic lactate metabolism in this strain.

The acute diarrhoeal disease cholera is caused by the aquatic pathogen Vibrio cholerae upon ingestion of contaminated food or water by the human host. The mechanisms by which V. cholerae is able to persist and survive in the host and aquatic environments have been studied for years; however, little is known about the factors involved in the adaptation or response of V. cholerae transitioning between these two environments. The transition from bacillary to coccoid morphology is thought to be one mechanism of survival that V. cholerae uses in response to environmental stress. Coccoid morphology has been observed for V. cholerae while in a viable but non-culturable (VBNC) state, during times of nutrient limitation, and in the water-diluted stool of cholera-infected patients. In this study we sought conditions to study the coccoid morphology of V. cholerae, and found that coccoid-shaped cells can express and produce the virulence factor toxin co-regulated pilus (TCP) and are able to colonize the infant mouse to the same extent as bacillus-shaped cells. This study suggests that TCP may be one factor that V. cholerae utilizes for adaptation and survival during the transition between the host and the aquatic environment.