- Volume 158, Issue 9, 2012

Fremyella diplosiphon modifies the pigment composition of its major light-harvesting complexes, i.e. phycobilisomes, and cell and filament morphology according to ambient light quality in a process termed complementary chromatic adaptation (CCA). The cells are red in colour and rectangular shaped, and filaments are longer under green light (GL), in contrast with blue-green, spherical cells and shorter filaments under red light (RL). In this study, we report that wild-type (WT) UTEX 481 and WT-pigmented, shortened filament strain SF33 of F. diplosiphon accumulate reactive oxygen species (ROS) under both GL and RL, with the level of oxidative stress being higher under RL as compared with GL. During CCA, higher levels of ROS under RL are correlated with the RL-specific spherical cell shape and filament fragmentation – cells exhibiting elevated levels of ROS under RL have reduced cell length, yet the width of cells is not affected. Addition of ascorbic acid to RL-grown cultures resulted in lower ROS levels and a concomitant shift to GL-associated cellular morphology, i.e. an increased cell length. This observation identifies an RL-dependent oxidative-stress-mediated regulation of morphogenesis in a bacterial system. Spherical cell morphology may result from ROS-dependent changes in the cell membrane integrity or cell wall loosening and associated cell expansion.

The locus of enterocyte effacement (LEE) is a 35 kb pathogenicity island involved in attaching and effacing (A/E) Escherichia coli enteric infection. The LEE is organized into five large transcriptional operons (LEE1–LEE5) and a few bi- and monocistronic instances. The LEE5 operon co-transcribes three genes, tir-cesT-eae, although cesT can be transcribed in a separate mRNA from its own proximal promoter. The role of this separate promoter is not understood. In this study we characterized promoter activity for the type III secretion chaperone gene cesT. The cesT promoter, cesTp, has features consistent with σ70 promoters that contain an extended −10 element. This was experimentally confirmed by mutations that altered cesTp activity. In stark contrast to LEE2–5 transcriptional operons, cesTp did not require the master regulator Ler for transcriptional activity. Moreover, cesTp activity was not dependent on the presence of GrlA or GrlR, two regulators associated with LEE gene expression. A cesTp–lux fusion was used in real-time assays and demonstrated initial cesTp activity that occurred before LEE5 promoter activity, which ensued after 120 min. cesTp was shown to be active during in vitro infection of HT-29 colonic epithelial cells. Inactivation of cesTp reduced CesT protein levels at early growth time points. These data indicate a Ler-, GrlA- and GrlR-independent activity for cesTp. We suggest that A/E pathogenic E. coli have evolved a mechanism to ready the cell for CesT protein expression in support of infection prior to Ler- and GrlA-related activities.

The poultry disease coccidiosis, caused by infection with Eimeria spp. apicomplexan parasites, is responsible for enormous economic losses to the global poultry industry. The rapid increase of resistance to therapeutic agents, as well as the expense of vaccination with live attenuated vaccines, requires the development of new effective treatments for coccidiosis. Because of their key regulatory function in the eukaryotic cell cycle, cyclin-dependent kinases (CDKs) are prominent drug targets. The Eimeria tenella CDC2-related kinase 2 (EtCRK2) is a validated drug target that can be activated in vitro by the CDK activator XlRINGO (Xenopus laevis rapid inducer of G2/M progression in oocytes). Bioinformatics analyses revealed four putative E. tenella cyclins (EtCYCs) that are closely related to cyclins found in the human apicomplexan parasite Plasmodium falciparum. EtCYC3a was cloned, expressed in Escherichia coli and purified in a complex with EtCRK2. Using the non-radioactive time-resolved fluorescence energy transfer (TR-FRET) assay, we demonstrated the ability of EtCYC3a to activate EtCRK2 as shown previously for XlRINGO. The EtCRK2/EtCYC3a complex was used for a combined in vitro and in silico high-throughput screening approach, which resulted in three lead structures, a naphthoquinone, an 8-hydroxyquinoline and a 2-pyrimidinyl-aminopiperidine-propane-2-ol. This constitutes a promising starting point for the subsequent lead optimization phase and the development of novel anticoccidial drugs.

The maintenance of cellular calcium homeostasis is associated with cellular signalling transduction and the functions of many membrane compartments, especially endoplasmic reticulum (ER) function. ER-localized proteins that serve to maintain ER and cellular calcium homeostasis in Candida albicans are still unclear. In this study, Spf1, the putative C. albicans homologue of the Saccharomyces cerevisiae ER-localized P-type calcium ATPase ScSpf1, was investigated for its roles in cellular calcium homeostasis, hyphal development and virulence. We constructed an Spf1 null mutant which showed decreased vegetative growth rate and hypersensitivity to EGTA, high-level calcium and antifungal drugs. Similar to treatments of ER stress agents, deletion of SPF1 stimulated calcium influx in the presence of FK506, resulting in an increase in cellular calcium contents, and induced expression of the calcium-dependent response elements gene CCH1, which is essential for the cell calcium survival pathway. Moreover, the spf1 null mutant had defects in hyphal development and biofilm formation, and was severely attenuated in virulence. These findings provided phenotypic evidence supporting roles for Spf1 in the maintenance of cellular calcium homeostasis, ER stress responses, hyphal development, biofilm formation and virulence in C. albicans.

Chemoreceptors sense environmental stimuli and transmit the information to the flagellar motors via a histidine kinase that controls the phosphorylation level of the effector protein CheY. The cytoplasmic domain of chemoreceptors consists of a long α-helical hairpin that forms, in the dimer, a coiled-coil four-helix bundle. Even though the sequence and general structure of the cytoplasmic domain are strongly conserved within Eubacteria and Archaea, the total length of the α-helical hairpin is variable and defines seven classes of chemoreceptors. In this work we assessed the functional properties of a Pseudomonas receptor when assembled in signalling complexes with Escherichia coli proteins. Our results show that the foreign receptor does not confer fully chemotactic abilities upon E. coli cells, but is able to form active ternary complexes that respond to the specific stimuli by modulating the activity of the associated kinase. The observed responses are subject to adaptation, depending on the presence of the methylation enzymes CheR and/or CheB. The ability of foreign receptors to signal through signalling complexes with non-cognate proteins would allow the use of the well-studied E. coli system to reveal the detection specificity of uncharacterized chemoreceptors from other micro-organisms.

Although arsenic is notoriously poisonous to life, its utilization in therapeutics brings many benefits to human health, so it is therefore essential to discover the molecular mechanisms underlying arsenic stress responses in eukaryotic cells. Aiming to determine the contribution of Ca2+ signalling pathways to arsenic stress responses, we took advantage of the use of Saccharomyces cerevisiae as a model organism. Here we show that Ca2+ enhances the tolerance of the wild-type and arsenic-sensitive yap1 strains to arsenic stress in a Crz1-dependent manner, thus providing the first evidence that Ca2+ signalling cascades are involved in arsenic stress responses. Moreover, our results indicate that arsenic shock elicits a cytosolic Ca2+ burst in these strains, without the addition of exogenous Ca2+ sources, strongly supporting the notion that Ca2+ homeostasis is disrupted by arsenic stress. In response to an arsenite-induced increase of Ca2+ in the cytosol, Crz1 is dephosphorylated and translocated to the nucleus, and stimulates CDRE-driven expression of the lacZ reporter gene in a Cnb1-dependent manner. The activation of Crz1 by arsenite culminates in the induction of the endogenous genes PMR1, PMC1 and GSC2. Taken together, these data establish that activation of Ca2+ signalling pathways and the downstream activation of the Crz1 transcription factor contribute to arsenic tolerance in the eukaryotic model organism S. cerevisiae.

Vibrio parahaemolyticus pandemic serotype O3 : K6 causes acute gastroenteritis, wound infections and septicaemia in humans. This organism encodes two type III secretion systems (T3SS1 and T3SS2); host-cell cytotoxicity has been attributed to T3SS1. Synthesis and secretion of T3SS1 proteins is positively regulated by ExsA, which is presumptively regulated by the ExsCDE pathway, similar to Pseudomonas aeruginosa. Herein we deleted the putative exsE from V. parahaemolyticus and found constitutive expression of the T3SS1 in broth culture as expected. More importantly, however, in a cell culture model, the ΔexsE strain was unable to induce cytotoxicity, as measured by release of lactate dehydrogenase (LDH), or autophagy, as measured by LC3 conversion. This is markedly different from P. aeruginosa, where deletion of exsE has no effect on host-cell cytolysis. Swarming and cytoadhesion were reduced for the deletion mutant and could be recovered along with T3SS1-induced HeLa cell cytotoxicity by in cis expression of exsE in the ΔexsE strain. Loss of adhesion and swarming motility was associated with the loss of flagella biogenesis in the exsE-deficient strain. Mouse mortality was unaffected by the deletion of exsE compared with a wild-type control, suggesting that additional adhesins are important for intoxication in vivo. Based on these data, we conclude that ExsE contributes to the negative regulation of T3SS1 and, in addition, contributes to regulation of an adherence phenotype that is requisite for translocation of effector proteins into HeLa cells.

Burkholderia cenocepacia is a member of the Burkholderia cepacia complex (Bcc), a group of Gram-negative opportunistic pathogens that cause severe lung infections in patients with cystic fibrosis and display extreme intrinsic resistance to antibiotics, including antimicrobial peptides. B. cenocepacia BCAL2157 encodes a protein homologous to SuhB, an inositol-1-monophosphatase from Escherichia coli, which was suggested to participate in post-transcriptional control of gene expression. In this work we show that a deletion of the suhB-like gene in B. cenocepacia (ΔsuhBBc ) was associated with pleiotropic phenotypes. The ΔsuhBBc mutant had a growth defect manifested by an almost twofold increase in the generation time relative to the parental strain. The mutant also had a general defect in protein secretion, motility and biofilm formation. Further analysis of the type II and type VI secretion systems (T2SS and T6SS) activities revealed that these secretion systems were inactive in the ΔsuhBBc mutant. In addition, the mutant exhibited increased susceptibility to polymyxin B but not to aminoglycosides such as gentamicin and kanamycin. Together, our results demonstrate that suhBBc deletion compromises general protein secretion, including the activity of the T2SS and the T6SS, and affects polymyxin B resistance, motility and biofilm formation. The pleiotropic effects observed upon suhBBc deletion demonstrate that suhBBc plays a critical role in the physiology of B. cenocepacia.

In living organisms, copper (Cu) contributes to essential functions but at high concentrations it may elicit toxic effects. Cu-tolerant yeast strains are of relevance for both biotechnological applications and studying physiological and molecular mechanisms involved in stress resistance. One way to obtain tolerant strains is to exploit experimental methods that rely on the principles of natural evolution (evolutionary engineering) and allow for the development of complex phenotypic traits. However, in most cases, the molecular and physiological basis of the phenotypic changes produced have not yet been unravelled. We investigated the determinants of Cu resistance in a Saccharomyces cerevisiae strain that was evolved to tolerate up to 2.5 g CuSO4 l−1 in the culture medium. We found that the content of intracellular Cu and the expression levels of several genes encoding proteins involved in Cu metabolism and oxidative stress response were similar in the Cu-tolerant (evolved) and the Cu-sensitive (non-evolved) strain. The major difference detected in the two strains was the copy number of the gene CUP1, which encodes a metallothionein. In evolved cells, a sevenfold amplification of CUP1 was observed, accounting for its strongly and steadily increased expression. Our results implicate CUP1 in protection of the evolved S. cerevisiae cells against Cu toxicity. In these cells, robustness towards Cu is stably inheritable and can be reproducibly selected by controlling environmental conditions. This finding corroborates the effectiveness of laboratory evolution of whole cells as a tool to develop microbial strains for biotechnological applications.

The Clp/HSP100 family of molecular chaperones is ubiquitous in both prokaryotes and eukaryotes. These proteins play important roles in refolding, disaggregating and degrading proteins damaged by stress. As a subclass of the Clp/HSP100 family, ClpB has been shown to be involved in various stress responses as well as other functions in bacteria. In the present study, we investigated the role of a predicted ClpB-encoding gene, MXAN5092, in the stress response during vegetative growth and development of Myxococcus xanthus. Transcriptional analysis confirmed induction of this clpB homologue under different stress conditions, and further phenotypic analysis revealed that an in-frame deletion mutant of MXAN5092 was more sensitive to various stress treatments than the wild-type strain during vegetative growth. Moreover, the absence of the MXAN5092 gene resulted in decreased heat tolerance of myxospores, indicating the involvement of this clpB homologue in the stress response during the development of myxospores. The M. xanthus recombinant ClpB (MXAN5092) protein also showed a general chaperone activity in vitro. Overall, our genetic and phenotypic analysis of the predicted ATP-dependent chaperone protein ClpB (MXAN5092) demonstrated that it functions as a chaperone protein and plays an important role in cellular stress tolerance during both vegetative growth and development of M. xanthus.

Only about half of bacterial species use an asparaginyl-tRNA synthetase (AsnRS) to attach Asn to its cognate tRNAAsn. Other bacteria, including the human pathogen Moraxella catarrhalis, a causative agent of otitis media, lack a gene encoding AsnRS, and form Asn-tRNAAsn by an indirect pathway catalysed by two enzymes: first, a non-discriminating aspartyl-tRNA synthetase (ND-AspRS) catalyses the formation of aspartyl-tRNAAsn (Asp-tRNAAsn); then, a tRNA-dependent amidotransferase (GatCAB) transamidates this ‘incorrect’ product into Asn-tRNAAsn. As M. catarrhalis has a Gln-tRNA synthetase, its GatCAB functions as an Asp-tRNAAsn amidotransferase. This pathogen rapidly evolved to about 90 % ampicillin resistance worldwide by insertion of a bro-1 β-lactamase gene within the gatCAB operon. Comparison of the GatCAB subunits from bro-1 β-lactamase-positive and bro-negative strains showed that the laterally transferred bro-1 gene, inserted into the gatCAB operon, affected the C-terminal sequence of GatA. The identity between the C-terminal sequences of GatAwt (residues 479–491) and of GatABRO-1 (residues 479–492) was about 36 %, whereas the rest of the GatA sequence was relatively conserved. The characterization of these two distinct GatCABs as well as the hybrid GatCAB containing GatA(1–478)wt(479–492)BRO-1 and truncated GatCAB enzymes of M. catarrhalis showed that the substitution in GatAwt of residues 479–492 of GatABRO-1 causes increased specificity for glutamine, and decreased specificity for Asp-tRNAAsn in the transamidation reaction. We conclude that the bro gene insertion has altered the kinetic parameters of Asp-tRNAAsn amidotransferase, and we propose a model for gatA evolution after the insertion of bro-1 at the carboxyl end of gatA.

Non-typable Haemophilus influenzae (NTHi) is a common commensal of the human nasopharynx, but causes opportunistic infection when the respiratory tract is compromised by infection or disease. The ability of NTHi to invade epithelial cells has been described, but the underlying molecular mechanisms are poorly characterized. We previously determined that NTHi promotes phosphorylation of the serine-threonine kinase Akt in A549 human lung epithelial cells, and that Akt phosphorylation and NTHi cell invasion are prevented by inhibition of phosphoinositide 3-kinase (PI3K). Because PI3K-Akt signalling is associated with several host cell networks, the purpose of the current study was to identify eukaryotic molecules important for NTHi epithelial invasion. We found that inhibition of Akt activity reduced NTHi internalization; differently, bacterial entry was increased by phospholipase Cγ1 inhibition but was not affected by protein kinase inhibition. We also found that α5 and β1 integrins, and the tyrosine kinases focal adhesion kinase and Src, are important for NTHi A549 cell invasion. NTHi internalization was shown to be favoured by activation of Rac1 guanosine triphosphatase (GTPase), together with the guanine nucleotide exchange factor Vav2 and the effector Pak1. Also, Pak1 might be associated with inactivation of the microtubule destabilizing agent Op18/stathmin, to facilitate microtubule polymerization and NTHi entry. Conversely, inhibition of RhoA GTPase and its effector ROCK increased the number of internalized bacteria. Src and Rac1 were found to be important for NTHi-triggered Akt phosphorylation. An increase in host cyclic AMP reduced bacterial entry, which was linked to protein kinase A. These findings suggest that NTHi finely manipulates host signalling molecules to invade respiratory epithelial cells.

Enterohaemorrhagic Escherichia coli (EHEC) survives exposure to acute acid stress during gastric passage and progresses to colonize the large intestine. We previously reported that acid stress significantly increases host adhesion of EHEC O157 : H7 and is associated with a coincident upregulation of the expression of a putative adhesin gene, yadK. Further gene expression analysis now confirms that yadK is minimally transcribed under unstressed conditions and is significantly upregulated under acid stress. Immunoblotting with an anti-YadK polyclonal antiserum demonstrates that YadK protein is also upregulated after acid stress. Disruption of yadK results in loss of the acid-induced adhesion increase seen for wild-type EHEC to human epithelial cells in vitro and complementation in trans fully restores the acid-induced adhesion phenotype to the wild-type level. Significantly, no difference is observed in adhesion of the unstressed yadK mutant relative to wild-type, indicating that YadK does not play a role in adhesion of unstressed EHEC. Anti-YadK antiserum inhibits the acid-induced adhesion enhancement of EHEC but has no effect on adhesion of unstressed EHEC. There is no significant difference in the viability of either the unstressed or the acid-stressed yadK mutant relative to the similarly treated wild-type, suggesting that yadK is not involved in acid tolerance. These results provide persuasive evidence that YadK plays a significant role in the adhesion of acid-stressed EHEC to epithelial cells, and support a role for acid stress as a factor which may regulate bacteria–host attachment and lead to increased EHEC colonization and virulence.

Sporothrix (Sp.) schenckii is a pathogenic fungus that infects humans and animals, and is responsible for the disease named sporotrichosis. The cell wall of this fungus has glycoproteins with a high content of mannose and rhamnose units, which are synthesized by endoplasmic reticulum- and Golgi-localized glycosyltransferases. Little is known about the enzymic machinery involved in the synthesis of these oligosaccharides in Sp. schenckii, or the genes encoding these activities. This is in part because of the lack of an available genome sequence for this organism. Using a partial genomic DNA library we identified SsMNT1, whose predicted product has significant similarity to proteins encoded by members of the Saccharomyces (Sa.) cerevisiae KRE2/MNT1 gene family. In order to biochemically characterize the putative enzyme, SsMNT1 was heterologously expressed in the methylotrophic yeast Pichia pastoris. Recombinant SsMnt1 showed Mn2+-dependent mannosyltransferase activity and the ability to recognize as acceptors α-methyl mannoside, mannose, Man5GlcNAc2 oligosaccharide and a variety of mannobiosides. The characterization of the enzymic products generated by SsMnt1 revealed that the enzyme is an α1,2-mannosyltransferase that adds up to two mannose residues to the acceptor molecule. Functional complementation studies were performed in Sa. cerevisiae and Candida albicans mutants lacking members of the KRE2/MNT1 gene family, demonstrating that SsMnt1 is involved in both the N- and O-linked glycosylation pathways, but not in phosphomannan elaboration.

Here, we show that Corynebacterium glutamicum ATCC 13032 co-metabolizes formate when it is grown with glucose as the carbon and energy source. CO2 measurements during bioreactor cultivation and use of 13C-labelled formate demonstrated that formate is almost completely oxidized to CO2. The deletion of fdhF (cg0618), annotated as formate dehydrogenase (FDH) and located in a cluster of genes conserved in the family Corynebacteriaceae, prevented formate utilization. Similarly, deletion of fdhD (cg0616) resulted in the inability to metabolize formate and deletion of cg0617 markedly reduced formate utilization. These results illustrated that all three gene products are required for FDH activity. Growth studies with molybdate and tungstate indicated that the FDH from C. glutamicum ATCC 13032 is a molybdenum-dependent enzyme. The presence of 100 mM formate caused a 25 % lowered growth rate during cultivation of C. glutamicum ATCC 13032 wild-type in glucose minimal medium. This inhibitory effect was increased in the strains lacking FDH activity. Our data demonstrate that C. glutamicum ATCC 13032 possesses an FDH with a currently unknown electron acceptor. The presence of the FDH might help the soil bacterium C. glutamicum ATCC 13032 to alleviate growth retardation caused by formate, which is ubiquitously present in the environment.