- Volume 153, Issue 11, 2007

This study presents morphological and biochemical evidence of programmed cell death (PCD) in Entamoeba histolytica induced by exposure of trophozoites to the aminoglycoside antibiotic G418. Morphological characteristics of PCD, including cell shrinkage, reduced cellular volume, nuclear condensation, DNA fragmentation and vacuolization were observed, with preservation of trophozoite membrane integrity. PCD is orchestrated biochemically by alterations in intracellular ion fluxes. In G418-treated trophozoites, overproduction of reactive oxygen species (ROS), decreased intracellular K+, increased cytosolic calcium, and decreased intracellular pH levels were observed. However, externalization of phosphatidylserine was not detected. These results suggest that amoebae can undergo PCD under stress conditions, and that this PCD shares several properties with PCD reported in mammals and in a variety of unicellular organisms.

Light is an environmental factor that regulates pivotal processes in living organisms, and appropriate perception is key to adaptation to the environment. Blue light activates asexual reproduction in Trichoderma atroviride through transcription factors BLR-1 and BLR-2 which regulate light-responsive genes. Here, we show that blr-2 expression is a limiting factor for photo-perception and photo-transduction. Overexpression of blr-2 resulted in increased photoconidiation and stronger expression of light-induced genes. In contrast, overexpression of blr-1 resulted in reduced photoconidiaton and weaker expression of light-induced genes. blr-2 overexpression caused a marked reduction of growth when the fungus was grown under defined photoperiods, including a period of strong sensitivity to light, followed by a period of insensitivity. Long periods of incubation under this condition permitted recovery of a rhythmic growth similar to that of the wild-type. In addition, blr-2 expression is apparently regulated at the post-transcriptional level through the BLR proteins and its expression level is BLR-1-dependent even in the dark. Finally, we demonstrated that blr-2 overexpression caused higher sensitivity to blue light and we therefore propose that the preformation of BLR-1/BLR-2 complexes is key to adequate light perception in T. atroviride.

The production of several bacteriocins in lactic acid bacteria is regulated by inducer peptide pheromones that specifically interact with their cognate bacterial receptor. These peptide pheromones are between 19 and 27 aa long and contain a conserved (V/I)-X-X-X-F sequence followed by positively charged residues in the C-terminal domain. CbaX and EntF are peptide pheromones that share similarity and are involved in the production of carnobacteriocin A in Carnobacterium maltaromaticum LV17A and enterocins A and B in Enterococcus faecium CTC492, respectively. CbaX, EntF and two hybrids, CbaX : : EntF and EntF : : CbaX, were tested for pheromone activity in LV17A and CTC492. EntF and EntF : : CbaX only induced bacteriocin production in CTC492, whereas CbaX and CbaX : : EntF induced carnobacteriocin A production in LV17A and, at high concentrations, also cross-induced enterocin production in CTC492. Various peptide fragments of CbaX and EntF were made for further structure–function analysis. The C-terminal fragments, but not the N-terminal fragments, were able to effect bacteriocin induction. The 10-mer EntF(16–25), derived from the C-terminal domain of EntF, showed pheromone activity in LV17A. In contrast, the C-terminal 9-mer of CbaX, CbaX(16–24), inhibited pheromone activity in both LV17A and CTC492. EntF(16–25) and CbaX(16–24) differ by two amino acids. Changing either one of these abolished pheromone activity as well as the ability to inhibit pheromone activity. These results indicate that the C-terminal domain of these peptide pheromones interacts relatively non-specifically with the receptor, and that induction is greatly facilitated by the N-terminal domain that recognizes specifically its cognate receptor.

Alcohol dehydrogenase 1 (Adh1)p catalyses the conversion of acetaldehyde to ethanol, regenerating NAD+. In Saccharomyces cerevisiae, Adh1p is oxidatively modified during ageing and, consequently, its activity becomes reduced. To analyse whether maintaining this activity is advantageous for the cell, a yeast strain with an extra copy of the ADH1 gene (2×ADH1) was constructed, and the effects on chronological and replicative ageing were analysed. The strain showed increased survival in stationary phase (chronological ageing) due to induction of antioxidant enzymes such as catalase and superoxide dismutases. In addition, 2×ADH1 cells displayed an increased activity of silent information regulator 2 (Sir2)p, an NAD+-dependent histone deacetylase, due to a higher NAD+/NADH ratio. As a consequence, a 30 % extension in replicative life span was observed. Taken together, these results suggest that the maintenance of enzymes that participate in NAD+/NADH balancing is important to chronological and replicative life-span parameters.

The Saccharomyces cerevisiae UGA4 gene, which encodes the γ-aminobutyric acid (GABA) and δ-aminolaevulinic acid (ALA) permease, is well known to be regulated by the nitrogen source. Its expression levels are low in the presence of a rich nitrogen source but are higher when a poor nitrogen source is used. In addition, GABA can induce UGA4 expression when cells are grown with proline but not when they are grown with ammonium. Although vast amounts of evidence have been gathered about UGA4 regulation by nitrogen, little is known about its regulation by the carbon source. Using glucose and acetate as rich and poor carbon source respectively, this work aimed to shed light on hitherto unclear aspects of the regulation of this gene. In poor nitrogen conditions, cells grown with acetate were found to have higher UGA4 basal expression levels than those grown with glucose, and did not show UGA4 induction in response to GABA. Analysis of the expression and subcellular localization of the transcription factors that regulate UGA4 as well as partial deletions and site-directed mutations of the UGA4 promoter region suggested that there are two parallel pathways that act in regulating this gene by the carbon source. Furthermore, the results demonstrate the existence of a new factor operating in UGA4 regulation.

Global transcriptional responses to dehydration and rehydration were determined in Anabaena sp. PCC 7120. Nearly 300 genes were up- or downregulated during both dehydration and rehydration. While as many as 133 genes showed dehydration-specific downregulation, only 29 genes showed dehydration-specific upregulation. In contrast, while only 13 genes showed rehydration-specific downregulation, as many as 259 genes showed rehydration-specific upregulation. The genes upregulated during rehydration responded rapidly and transiently, whereas those upregulated during dehydration did so gradually and persistently. The expression of various genes involved in DNA repair, protein folding and NAD synthesis, as well as genes responding to nitrogen depletion and CO2 limitation, was upregulated during rehydration. Although no genes for transcriptional regulators showed dehydration-specific upregulation, eight showed rehydration-specific upregulation. Among them, two genes, ancrpB and alr0618, encode putative transcriptional activators of the cAMP receptor protein (CRP) family. DNA microarray analysis using gene disruptants revealed that AnCrpB and Alr0618 regulate the genes induced by nitrogen depletion and by CO2 limitation, respectively. We conclude that rehydration is a complex process in which the expression of certain genes, particularly those for metabolism, is dramatically induced.

Microcystis aeruginosa is one of the most prevalent bloom-forming cyanobacteria and has been the cause of increasing public health concern due to the production of hepatotoxins (microcystins). To investigate the genetic diversity, clonality and evolutionary genetic background with regard to the toxicity of M. aeruginosa, a multilocus sequence typing (MLST) scheme was developed, based on seven selected housekeeping loci (ftsZ, glnA, gltX, gyrB, pgi, recA and tpi). Analysis of a collection of 164 isolates from Japan and other countries identified 79 unique sequence types (STs), revealing a high level of genetic diversity (H=0.951). Although recombination between loci was indicated to be substantial by Shimodaira–Hasegawa (SH) tests, multilocus linkage disequilibrium analyses indicated that recombination between strains probably occurs at some frequency but not to the extent at which alleles are associated randomly, suggesting that the population structure of M. aeruginosa is clonal. Analysis of subsets of strains also indicated that the clonal population structure is maintained even in a local population. Phylogenetic analysis based on the concatenated sequences of seven MLST loci demonstrated that microcystin-producing genotypes are not monophyletic, providing further evidence for the gain and loss of toxicity during the intraspecific diversification of M. aeruginosa. However, toxic strains are genetically distinct from non-toxic strains in MLST allelic profiles, and it was also shown that non-toxic strains harbouring toxin genes fall into a single monophyletic clade, except for one case. These results suggest that the toxicity of M. aeruginosa is relatively stable in the short term, and therefore can be unequivocally characterized by MLST. The MLST scheme established here will be of great help for future detailed population genetic studies of M. aeruginosa.

In this study, the Enterobacteriaceae microbiota, including their diversity as well as the distribution of haemolytic and virulence gene-harbouring Escherichia coli of 56-day-old healthy piglets, was characterized. Both the composition and the diversity of Enterobacteriaceae populations varied considerably between individual pigs and intestinal sections. E. coli, Enterobacter cloacae, Citrobacter freundii and Klebsiella pneumoniae dominated the Enterobacteriaceae microbiota. However, mucosa-associated Enterobacteriaceae were scarce or in some cases undetectable. The majority of E. coli clones from the jejunum were also found in the colon, with up to 10 different E. coli clones in one intestinal section. Other Enterobacteriaceae species were represented by only one clone localized to one intestinal section. While several piglets did not harbour virulence gene-positive or haemolytic E. coli, such strains dominated intestinal sections of other animals. This study reveals that the diversity of intestinal Enterobacteriaceae is clearly individual. In general, Enterobacteriaceae do not appear to be a consistent fraction of the microbiota of the jejunum. High numbers of adherent bacteria do not appear to be essential for successful intestinal colonization, and E. coli clones do not necessarily colonize distinct intestinal sections based on the particular phylogenetic affiliation. Furthermore, dominance of haemolytic or virulence gene-positive E. coli does not correlate with disease. Finally, probiotic Enterococcus faecium feed supplementation does not affect the Enterobacteriaceae microbiota.

In the presence of ammonium ion, Nodularia spumigena strain AV1, a filamentous, heterocystous cyanobacterium isolated from the Baltic Sea, lost aerobic nitrogen-fixation activity while maintaining heterocyst frequency along the filaments. Real-time RT-PCR showed that the expression of nifH (encoding the dinitrogenase reductase component of the nitrogenase enzyme) was suppressed and the levels of NifH protein decreased dramatically in response to treatment with ammonium. On the other hand, ntcA (encoding the global nitrogen regulator in cyanobacteria) and hetR (the key regulatory gene in heterocyst differentiation) were expressed and their expression patterns were not affected by the treatment with ammonium. These data demonstrate that N. spumigena strain AV1 maintains heterocyst frequency along the filaments in the presence of ammonium and in the absence of detectable N2-fixation activity.

Comamonas sp. strain CNB-1, a chloronitrobenzene-degrading bacterium, was demonstrated to possess higher arsenate tolerance as compared with the mutant strain CNB-2. pCNB1, a plasmid harboured by CNB-1 but not CNB-2, contained the genetic cluster ars(RPBC)Com , which putatively encodes arsenate-resistance regulator, family II arsenate reductase, arsenite efflux pump and family I arsenate reductase, respectively, in Comamonas strain CNB-1. The arsC-negative Escherichia coli could gain arsenate resistance by transformation with arsPCom or arsCCom , indicating that these two genes might express functional forms of arsenate reductases. Intriguingly, when CNB-1 cells were exposed to arsenate, the transcription of arsPCom and arsCCom was measurable by RT-PCR, but only ArsP Com was detectable at protein level. To explore the proteins responding to arsenate stress, CNB-1 cells were cultured with and without arsenate and differential proteomics was carried out by two-dimensional PAGE (2-DE) and MALDI-TOF MS. A total of 31 differential 2-DE spots were defined upon image analysis and 23 proteins were identified to be responsive specifically to arsenate. Of these spots, 18 were unique proteins. These proteins were identified to be phosphate transporters, heat-shock proteins involved in protein refolding, and enzymes participating in carbon and energy metabolism.

Identification of short genes that encode peptides of fewer than 60 aa is challenging, both experimentally and in silico. As a consequence, the universe of these short coding sequences (CDSs) remains largely unknown, although some are acknowledged to play important roles in cell–cell communication, particularly in Gram-positive bacteria. This paper reports a thorough search for short CDSs across streptococcal genomes. Our bioinformatic approach relied on a combination of advanced intrinsic and extrinsic methods. In the first step, intrinsic sequence information (nucleotide composition and presence of RBSs) served to identify new short putative CDSs (spCDSs) and to eliminate the differences between annotation policies. In the second step, pseudogene fragments and false predictions were filtered out. The last step consisted of screening the remaining spCDSs for lines of extrinsic evidence involving sequence and gene-context comparisons. A total of 789 spCDSs across 20 complete genomes (19 Streptococcus and one Enterococcus) received the support of at least one line of extrinsic evidence, which corresponds to an average of 20 short CDSs per million base pairs. Most of these had no known function, and a significant fraction (31 %) are not even annotated as hypothetical genes in GenBank records. As an illustration of the value of this list, we describe a new family of CDSs, encoding very short hydrophobic peptides (20–23 aa) situated just upstream of some of the positive transcriptional regulators of the Rgg family. The expression of seven other short CDSs from Streptococcus thermophilus CNRZ1066 that encode peptides ranging in length from 41 to 56 aa was confirmed by real-time quantitative RT-PCR and revealed a variety of expression patterns. Finally, one peptide from this list, encoded by a gene that is not annotated in GenBank, was identified in a cell-envelope-enriched fraction of S. thermophilus CNRZ1066.

A new lactococcal plasmid, pDBORO, was isolated from the Lactococcus lactis subsp. lactis biovar diacetylactis strain DB0410. This plasmid is responsible for the sensitivity of DB0410 to the toxic pyrimidine analogue 5-fluoroorotate. The complete nucleotide sequence has been determined and amounts to 16 404 bp. Of 15 ORFs encountered, three were found to be insertion sequence (IS) elements, identified as two IS946 and one IS982. Two ORFs are incomplete due to the insertion of an IS element in their C-terminal region. Homologues for four ORFs were found in the IL1403 sequence: the copB gene, coding for a copper-potassium-transporting ATPase B, and the ysbA, ysbB and ysbC genes. The structural organization of the pDBORO replication region is highly similar to other theta-replicating plasmids in both the cis- (repA) and trans-acting (repB and orfX) sequences. By plasmid deletion analysis and molecular cloning, a single locus on pDBORO was found to confer sensitivity to 5-fluoroorotate. It was identified as ysbC, but renamed oroP in order to reflect its function. The oroP gene was found to be essential for the utilization of orotate as the sole pyrimidine source in a strain deficient in pyrimidine de novo synthesis. The amino acid sequence encoded by the ORF showed the characteristic features of a membrane protein. Therefore, oroP most probably encodes an orotate transporter. Surprisingly, homologues of oroP could be identified in the genomes of both L. lactis MG1363 and L. lactis IL1403 despite the fact that these strains were unable to significantly utilize orotate. Cloning of oroP in Escherichia coli and Bacillus subtilis showed that the orotate transport phenotype could be transformed to both organisms. The findings presented indicate that oroP can be used as a powerful, food-grade selection/counterselection marker in many different organisms.

Saccharomyces cerevisiae is unique among yeasts in its ability to grow rapidly in the complete absence of oxygen. S. cerevisiae is therefore an ideal eukaryotic model to study physiological adaptation to anaerobiosis. Recent transcriptome analyses have identified hundreds of genes that are transcriptionally regulated by oxygen availability but the relevance of this cellular response has not been systematically investigated at the key control level of the proteome. Therefore, the proteomic response of S. cerevisiae to anaerobiosis was investigated using metabolic stable-isotope labelling in aerobic and anaerobic glucose-limited chemostat cultures, followed by relative quantification of protein expression. Using independent replicate cultures and stringent statistical filtering, a robust dataset of 474 quantified proteins was generated, of which 249 showed differential expression levels. While some of these changes were consistent with previous transcriptome studies, many of the responses of S. cerevisiae to oxygen availability were, to our knowledge, previously unreported. Comparison of transcriptomes and proteomes from identical cultivations yielded strong evidence for post-transcriptional regulation of key cellular processes, including glycolysis, amino-acyl-tRNA synthesis, purine nucleotide synthesis and amino acid biosynthesis. The use of chemostat cultures provided well-controlled and reproducible culture conditions, which are essential for generating robust datasets at different cellular information levels. Integration of transcriptome and proteome data led to new insights into the physiology of anaerobically growing yeast that would not have been apparent from differential analyses at either the mRNA or protein level alone, thus illustrating the power of multi-level studies in yeast systems biology.

Informational genes such as those encoding rRNAs are related to transcription and translation, and are thus considered to be rarely subject to lateral gene transfer (LGT) between different organisms, compared to operational genes having metabolic functions. However, several lines of evidence have suggested or confirmed the occurrence of LGT of DNA segments encoding evolutionarily variable regions of rRNA genes between different organisms. In the present paper, we show, for the first time to our knowledge, that variable regions of the 18S rRNA gene are segmentally replaced by multiple copies of different sequences in a single strain of the green microalga Prototheca wickerhamii, resulting in at least 17 genotypes, nine of which were actually transcribed. Recombination between different 18S rRNA genes occurred in seven out of eight variable regions (V1–V5 and V7–V9) of eukaryotic small subunit (SSU) rRNAs. While no recombination was observed in V1, one to three different recombination loci were demonstrated for the other regions. Such segmental replacement was also implicated for helix H37, which is defined as V6 of prokaryotic SSU rRNAs. Our observations provide direct evidence for redundant recombination of an informational gene, which encodes a component of mature ribosomes, in a single strain of one organism.

bcr1 is a chromosomal ∼155 bp repeated element found uniquely and ubiquitously in the Bacillus cereus group of Gram-positive bacteria; it exhibits several features characteristic of mobile elements, including a variable distribution pattern between strains. Here, highly similar bcr1 elements in non-conserved genomic loci are identified in a set of closely related B. cereus and Bacillus thuringiensis strains near the Bacillus anthracis phylogenetic cluster. It is also shown that bcr1 may be present on small RNA transcripts in the 100–400 bp size range. In silico folding of bcr1 at the RNA level indicated that transcripts may form a double-hairpin-like structure predicted to have high structural stability. A functional role of bcr1 at the RNA level is supported by multiple cases of G–U base-pairing, and compensatory mutations maintaining structural stability of the RNA fold. In silico folding at the DNA level produced similar predicted structures, with the potential to form a cruciform structure at open DNA complexes. The predicted structural stability was greater for bcr1 elements showing high sequence identities to bcr1 elements in non-conserved chromosomal loci in other strains, relative to other bcr1 copies. bcr1 mobility could thus be dependent on the formation of a stable DNA or RNA intermediate. Furthermore, bcr1 elements potentially encoding structurally stable and less stable transcripts were phylogenetically intermixed, indicating that loss of bcr1 mobility may have occurred multiple times during evolution. Repeated elements with similar features in other bacteria have been shown to provide functions such as mRNA stabilization, transcription termination and/or promoter function. Similarly, bcr1 may constitute a mobile element which occasionally gains a function when it enters an appropriate chromosomal locus.

Helicobacter hepaticus, a causative agent of chronic hepatitis and hepatocellular carcinoma in mice, possesses a hydrogenase and a urease, both of which are nickel-containing enzymes. Analysis of the genome sequence of H. hepaticus revealed a full set of accessory genes which are required for the nickel maturation of each enzyme in other micro-organisms. Erythromycin-resistant mutants were constructed in four of these genes, hypA, hypB, ureE and ureG. Controls for polar effect were provided for hypA or hypB mutants by disrupting each gene located immediately downstream, i.e. hp0809 or hypC, respectively. Urease and hydrogenase activities were determined for each strain with or without supplemented nickel in the medium. As expected, the ureE and the ureG mutants had negligible urease activity, but they retained normal levels of hydrogenase activity. Urease levels could not be increased by the addition of nickel to the medium. The H. hepaticus hypA and hypB strains were deficient in both urease and hydrogenase activities, suggesting that both gene products act in a similar fashion as their counterparts in H. pylori. However, in contrast with the analogous mutants of H. pylori, the addition of nickel into the growth medium failed to restore either urease or hydrogenase enzyme levels in the H. hepaticus hypA or hypB mutants, indicating a probably unique role for these genes in the mouse liver pathogen.

Based on their localization at the boundary of the bacterial cell and its environment, outer-membrane proteins (Omps) are important determinants for interaction of bacteria with their host cell. Therefore, they can be considered as important determinants for virulence. Looking for Legionella pneumophila Omps potentially involved in virulence, we identified a gene encoding a homologue of the plasminogen activator (Pla) of Yersinia pestis. Pla belongs to the class of omptins, a family of surface proteases/adhesins that exhibit different virulence-associated functions. In this report we describe the cloning and identification of the plasminogen activator homologue Lpa of L. pneumophila and demonstrate its outer-membrane localization. Transcriptional analysis of the Lpa region revealed expression of the gene in both exponential and stationary growth phase and showed that transcription of the lpa gene is directed by its own promoter. We also show, to our knowledge for the first time, that L. pneumophila has the capacity to convert plasminogen into plasmin by the action of the outer-membrane Lpa protein.

The Agrobacterium tumefaciens VirB/D4 type IV secretion system (T4SS) mediates the transfer of single-stranded DNA and protein virulence factors into plant cells, and also determines the assembly of the T-pilus, which is believed to play a role in host recognition. The T-pilus is composed of the major component VirB2 and the minor component VirB5. Using immuno-electron microscopy we detected the major component VirB2 along the entire length of detached T-pili, but not on cell-bound T-pili or on the cell surface. In contrast, the minor T-pilus component VirB5 was detected on the tips of cell-bound T-pili as well as on the ends of detached T-pili and on the cell surface. To gain further insights into the role of VirB5 we introduced changes at its C terminus. C-terminal deletions of up to four amino acids and alanine replacements did not abolish T-pilus formation and incorporation of the VirB5 variants at the tip, although they did impact the length of T-pili. Also, these changes differentially affected the ability of the T4SS to transfer DNA into plant and bacterial recipients, suggesting differential effects on host-cell specificity. The data presented here suggest that VirB5 localizes at the T-pilus tip, and provide novel insights into its role during the type IV secretion process.

Enterotoxigenic Escherichia coli (ETEC) causes enterotoxin-induced diarrhoea and significant mortality. The molecular mechanisms underlying how the heat-labile enterotoxin (LT) is secreted during infection are poorly understood. ETEC produce outer-membrane vesicles (OMVs) containing LT that are endocytosed into host cells. Although OMV production and protein content may be a regulated component of ETEC pathogenesis, how LT loading into OMVs is regulated is unknown. The LeoA protein plays a role in secreting LT from the bacterial periplasm. To begin to understand the function of LeoA and its role in ETEC H10407 pathogenesis, a site-directed mutant lacking the putative GTP-binding domain was constructed. The ability of wild-type and mutant LeoA to hydrolyse GTP in vitro was quantified. This domain was found to be responsible for GTP binding; it is important to LeoA's function in LT secretion, and may play a modest role in the formation and protein content of OMVs. Deletion of leoA reduced the abundance of OmpX in outer-membrane protein preparations and of LT in OMVs. Immunoprecipitation experiments revealed that LeoA interacts directly with OmpA, but that the GTP-binding domain is non-essential for this interaction. Deletion of leoA rendered ETEC H10407 non-motile, through apparent periplasmic accumulation of FliC.

Mycoplasma hyopneumoniae, the causative agent of swine enzootic pneumonia, colonizes the cilia of swine lungs, causing ciliostasis and cell death. M. hyopneumoniae is a component of the porcine respiratory disease complex (PRDC) and is especially problematic for the finishing swine industry, causing the loss of hundreds of millions of dollars in farm revenues worldwide. For successful infection, M. hyopneumoniae must effectively resist oxidative stresses due to the release of oxidative compounds from neutrophils and macrophages during the host's immune response. However, the mechanism that M. hyopneumoniae uses to avert the host response is still unclear. To gain a better understanding of the transcriptional responses of M. hyopneumoniae under oxidative stress, cultures were grown to early exponential phase and exposed to 0.5 % hydrogen peroxide for 15 min. RNA samples from these cultures were collected and compared to RNA samples from control cultures using two-colour PCR-based M. hyopneumoniae microarrays. This study revealed significant downregulation of important glycolytic pathway genes and gene transcription proteins, as well as a protein known to activate oxidative stressor cascades in neutrophils. Sixty-nine per cent of the upregulated genes were hypothetical with no known function. This study has also revealed significantly differentially expressed genes common to other environmental stress responses, indicating that further investigation of universal stress response genes of M. hyopneumoniae is merited.