- Volume 160, Issue 2, 2014

Pyocins are toxic proteins produced by some strains of Pseudomonas aeruginosa that are lethal for related strains of the same species. Some soluble pyocins (S2, S3 and S4) were previously shown to use the pyoverdine siderophore receptors to enter the cell. The P. aeruginosa PAO1 pore-forming pyocin S5 encoding gene (PAO985) was cloned into the expression vector pET15b, and the affinity-purified protein product tested for its killing activity against different P. aeruginosa strains. The results, however, did not show any correlation with a specific ferripyoverdine receptor. To further identify the S5 receptor, transposon mutants were generated. Pooled mutants were exposed to pyocin S5 and the resistant colonies growing in the killing zone were selected. The majority of S5-resistant mutants had an insertion in the fptA gene encoding the receptor for the siderophore pyochelin. Complementation of an fptA transposon mutant with the P. aeruginosa fptA gene in trans restored the sensitivity to S5. In order to define the receptor-binding domain of pyocin S5, two hybrid pyocins were constructed containing different regions from pyocin S5 fused to the C-terminal translocation and DNase killing domains of pyocin S2. Only the protein containing amino acid residues 151 to 300 from S5 showed toxicity, indicating that the pyocin S5 receptor-binding domain is not at the N-terminus of the protein as in other S-type pyocins. Pyocin S5 was, however, unable to kill Burkholderia cenocepacia strains producing a ferripyochelin FptA receptor, nor was the B. cenocepacia fptA gene able to restore the sensitivity of the resistant fptA mutant P. aeruginosa strain.

Haem-dependent catalase is an antioxidant enzyme that degrades H2O2, producing H2O and O2, and is common in aerobes. Catalase is present in some strictly anaerobic methane-producing archaea (methanogens), but the importance of catalase to the antioxidant system of methanogens is poorly understood. We report here that a survey of the sequenced genomes of methanogens revealed that the majority of species lack genes encoding catalase. Moreover, Methanosarcina acetivorans is a methanogen capable of synthesizing haem and encodes haem-dependent catalase in its genome; yet, Methanosarcina acetivorans cells lack detectable catalase activity. However, inducible expression of the haem-dependent catalase from Escherichia coli (EcKatG) in the chromosome of Methanosarcina acetivorans resulted in a 100-fold increase in the endogenous catalase activity compared with uninduced cells. The increased catalase activity conferred a 10-fold increase in the resistance of EcKatG-induced cells to H2O2 compared with uninduced cells. The EcKatG-induced cells were also able to grow when exposed to levels of H2O2 that inhibited or killed uninduced cells. However, despite the significant increase in catalase activity, growth studies revealed that EcKatG-induced cells did not exhibit increased tolerance to O2 compared with uninduced cells. These results support the lack of catalase in the majority of methanogens, since methanogens are more likely to encounter O2 rather than high concentrations of H2O2 in the natural environment. Catalase appears to be a minor component of the antioxidant system in methanogens, even those that are aerotolerant, including Methanosarcina acetivorans. Importantly, the experimental approach used here demonstrated the feasibility of engineering beneficial traits, such as H2O2 tolerance, in methanogens.

Group G Streptococcus (GGS) is a human bacterial pathogen expressing surface proteins FOG and protein G (PG) which interact with several host defence systems, including the complement and contact systems. Selected reaction monitoring mass spectrometry, electron microscopy and protein binding assays were used to track the amounts of FOG and PG intracellularly and on the bacterial surface during different phases of growth. Large and increasing amounts of PG were present on the surface in the stationary growth phase, and this was due to de novo production. In contrast, the amount of FOG did not change substantially during this phase. Apart from PG, a number of housekeeping proteins also increased in abundance in the stationary phase. These results show that GGS protein production is active during the stationary phase and that the bacteria actively remodel their surface and enter a less pro-inflammatory state in this phase.

The chromate ion transporter (CHR) superfamily comprises transporters that confer chromate resistance by extruding toxic chromate ions from cytoplasm. Burkholderia xenovorans strain LB400 has been reported to encode six CHR homologues in its multireplicon genome. We found that strain LB400 displays chromate-inducible resistance to chromate. Susceptibility tests of Escherichia coli strains transformed with cloned B. xenovorans chr genes indicated that the six genes confer chromate resistance, although under different growth conditions, and suggested that expression of chr genes is regulated by sulfate. Expression of chr genes was measured by quantitative reverse transcription-PCR (RT-qPCR) from total RNA of B. xenovorans LB400 grown under different concentrations of sulfate and exposed or not to chromate. The chr homologues displayed distinct expression levels, but showed no significant differences in transcription under the various sulfate concentrations tested, indicating that sulfate does not regulate chr gene expression in B. xenovorans. The chrA2 gene, encoded in the megaplasmid, was the only chr gene whose expression was induced by chromate and it was shown to constitute the chromate-responsive chrBACF operon. These data suggest that this determinant is mainly responsible for the B. xenovorans LB400 chromate resistance phenotype.

Cyanobacteria are photoautotrophic prokaryotes that occur in highly variable environments. Protein phosphorylation is one of the most widespread means to adjust cell metabolism and gene expression to the demands of changing growth conditions. Using a 2D gel electrophoresis-based approach and a phosphoprotein-specific dye, we investigated the protein phosphorylation pattern in cells of the model cyanobacterium Synechocystis sp. strain PCC 6803. The comparison of gels stained for total and phosphorylated proteins revealed that approximately 5 % of the protein spots seemed to be phosphoproteins, from which 32 were identified using MALDI-TOF MS. For eight of them the phosphorylated amino acid residues were mapped by subsequent mass spectrometric investigations of isolated phosphopeptides. Among the phosphoproteins, we found regulatory proteins, mostly putative anti-sigma factor antagonists, and proteins involved in translation. Moreover, a number of enzymes catalysing steps in glycolysis or the Calvin–Benson cycle were found to be phosphorylated, implying that protein phosphorylation might represent an important mechanism for the regulation of the primary carbon metabolism in cyanobacterial cells.

Streptococcus sanguinis is a Gram-positive bacterium that is indigenous to the oral cavity. S. sanguinis, a primary colonizer of the oral cavity, serves as a tether for the attachment of other oral pathogens. The colonization of microbes on the tooth surface forms dental plaque, which can lead to the onset of periodontal disease. We examined a comprehensive mutant library to identify genes related to cellular chain length and morphology using phase-contrast microscopy. A number of hypothetical genes related to the cellular chain length were identified in this study. Genes related to the cellular chain length were analysed along with clusters of orthologous groups (COG) for gene functions. It was discovered that the highest proportion of COG functions related to cellular chain length was ‘cell division and chromosome separation’. However, different COG functions were also found to be related with altered cellular chain length. This suggested that different genes related with multiple mechanisms contribute to the cellular chain length in S. sanguinis SK36.

The filamentous fungus Aspergillus niger is an industrially exploited protein expression platform, well known for its capacity to secrete high levels of proteins. To study the process of protein secretion in A. niger, we established a GFP-v-SNARE reporter strain in which the trafficking and dynamics of secretory vesicles can be followed in vivo. The biological role of putative A. niger orthologues of seven secretion-specific genes, known to function in key aspects of the protein secretion machinery in Saccharomyces cerevisiae, was analysed by constructing respective gene deletion mutants in the GFP-v-SNARE reporter strain. Comparison of the deletion phenotype of conserved proteins functioning in the secretory pathway revealed common features but also interesting differences between S. cerevisiae and A. niger. Deletion of the S. cerevisiae Sec2p orthologue in A. niger (SecB), encoding a guanine exchange factor for the GTPase Sec4p (SrgA in A. niger), did not have an obvious phenotype, while SEC2 deletion in S. cerevisiae is lethal. Similarly, deletion of the A. niger orthologue of the S. cerevisiae exocyst subunit Sec3p (SecC) did not result in a lethal phenotype as in S. cerevisiae, although severe growth reduction of A. niger was observed. Deletion of secA, secH and ssoA (encoding SecA, SecH and SsoA the A. niger orthologues of S. cerevisiae Sec1p, Sec8p and Sso1/2p, respectively) showed that these genes are essential for A. niger, similar to the situation in S. cerevisiae. These data demonstrate that the orchestration of exocyst-mediated vesicle transport is only partially conserved in S. cerevisiae and A. niger.

Giardia trophozoites differentiate into infectious cysts (encystment) in response to physiological stimuli; encystment is crucial for Giardia’s transmission, survival and pathogenesis. In vitro, Giardia encysts when bile sequesters lipids necessary for this lipid auxotroph, and in vivo they encyst to infect new hosts. In this study, we investigated, for the first time, commitment to encystment in Giardia using both molecular and cellular techniques. We show that after 3–6 h in inducing conditions, encysting trophozoites continue to encyst regardless of whether the inducing stimulus remains. We propose that a trophozoite’s inability to revert to a growing or dividing trophozoite represents a commitment to encystment. The onset of commitment correlated with the appearance of encystment specific vesicles (ESVs) and encystment specific protein synthesis. These observations suggest the involvement of regulatory pathways with the ability to ‘remember’ a transient signal long after its removal; a property that enables encysting trophozoites to complete the encystment process should the unfavourable triggering condition(s) change. The ability to form cysts in response to transient signals or, as we have highlighted in this paper, the ability of a small percentage of the population to form cysts without an inducer is vital for the maintenance of infection within populations.

Amphibacillus xylanus grows at the same rate and with the same cell yield under aerobic and anaerobic conditions. Under aerobic conditions, it exhibits vigorous oxygen consumption in spite of lacking a respiratory system and haem catalase. To understand the adaptive response of A. xylanus to oxidative stresses, a genomic analysis of A. xylanus was conducted. The analysis showed that A. xylanus has the genes of four metabolic systems: two pyruvate metabolic pathways, a glycolytic metabolic pathway and an NADH oxidase (Nox)–AhpC (Prx) system. A transcriptional study confirmed that A. xylanus has these metabolic systems. Moreover, genomic analysis revealed the presence of two genes for NADH oxidase (nox1 and nox2), both of which were identified in the transcriptional analysis. The nox1 gene in A. xylanus was highly expressed under normal aerobic conditions but that of nox2 was not. A purification study of NADH oxidases indicated that the gene product of nox1 is a primary metabolic enzyme responsible for metabolism of both oxygen and reactive oxygen species. A. xylanus was successfully grown under forced oxidative stress conditions such as 0.1 mM H2O2, 0.3 mM paraquat and 80 % oxygen. Proteomic analysis revealed that manganese SOD, Prx, pyruvate dehydrogenase complex E1 and E3 components, and riboflavin synthase β-chain are induced under normal aerobic conditions, and the other proteins except the five aerobically induced proteins were not induced under forced oxidative stress conditions. Taken together, the present findings indicate that A. xylanus has a unique defence system against forced oxidative stress.

To survive, the entomopathogenic fungus Beauveria bassiana, which shows promise as a biocontrol agent for a variety of pests, including agricultural and forestry pests and vectors of human pathogens, must tailor gene expression to the particular pH of its environment. The pH response transcription factor gene BbPacC and its flanking sequence were cloned from this fungus. Quantitative reverse transcription (RT)-PCR revealed that it is highly induced by alkaline pH and salt stress, and the expression level achieved twice that of the housekeeping gene γ-actin. A microfluorometric assay indicated that the 1479 bp promoter region could activate the expression of enhanced green fluorescent protein (EGFP) under the same conditions. Truncation analysis showed that the 1479, 1274, 1040, 888 and 742 bp promoters have similar efficiencies in activating expression of β-glucuronidase (GUS). The GUS activities of corresponding transformants reached approximately 50 % that of those containing the strong constitutive promoter PtrpC. A truncation upstream at the –572 bp position (referenced to the translation start codon ATG), however, resulted in a significant loss of GUS activity. Both the upstream absences of the −502 and −387 bp positions caused almost complete loss of GUS activity. These results suggest that PPacC is an efficient, alkaline, and salt-inducible promoter, the core cis-elements are mainly located within the –742 to –502 bp region, and promoters equal to or longer than 742 bp may be feasible for regulating gene expression in response to an ambient pH or salt stress.

Burkholderia cepacia complex (Bcc) bacteria possess biotechnologically useful properties that contrast with their opportunistic pathogenicity. The rhizosphere fitness of Bcc bacteria is central to their biocontrol and bioremediation activities. However, it is not known whether this differs between species or between environmental and clinical strains. We investigated the ability of 26 Bcc strains representing nine different species to colonize the roots of Arabidopsis thaliana and Pisum sativum (pea). Viable counts, scanning electron microscopy and bioluminescence imaging were used to assess root colonization, with Bcc bacteria achieving mean (±sem) levels of 2.49±0.23×106 and 5.16±1.87×106 c.f.u. per centimetre of root on the A. thaliana and P. sativum models, respectively. The A. thaliana rhizocompetence model was able to reveal loss of colonization phenotypes in Burkholderia vietnamiensis G4 transposon mutants that had only previously been observed in competition experiments on the P. sativum model. Different Bcc species colonized each plant model at different rates, and no statistical difference in root colonization was observed between isolates of clinical or environmental origin. Loss of the virulence-associated third chromosomal replicon (>1 Mb DNA) did not alter Bcc root colonization on A. thaliana. In summary, Bcc bacteria possess intrinsic root colonization abilities irrespective of their species or source. As Bcc rhizocompetence does not require their third chromosomal replicon, the possibility of using synthetic biology approaches to engineer virulence-attenuated biotechnological strains is tractable.

YadB and YadC are putative trimeric autotransporters present only in the plague bacterium Yersinia pestis and its evolutionary predecessor, Yersinia pseudotuberculosis. Previously, yadBC was found to promote invasion of epithelioid cells by Y. pestis grown at 37 °C. In this study, we found that yadBC also promotes uptake of 37 °C-grown Y. pestis by mouse monocyte/macrophage cells. We tested whether yadBC might be required for lethality of the systemic stage of plague in which the bacteria would be pre-adapted to mammalian body temperature before colonizing internal organs and found no requirement for early colonization or growth over 3 days. We tested the hypothesis that YadB and YadC function on ambient temperature-grown Y. pestis in the flea vector or soon after infection of the dermis in bubonic plague. We found that yadBC did not promote uptake by monocyte/macrophage cells if the bacteria were grown at 28 °C, nor was there a role of yadBC in colonization of fleas by Y. pestis grown at 21 °C. However, the presence of yadBC did promote recoverability of the bacteria from infected skin for 28 °C-grown Y. pestis. Furthermore, the gene for the proinflammatory chemokine CXCL1 was upregulated in expression if the infecting Y. pestis lacked yadBC but not if yadBC was present. Also, yadBC was not required for recoverability if the bacteria were grown at 37 °C. These findings imply that thermally induced virulence properties dominate over effects of yadBC during plague but that yadBC has a unique function early after transmission of Y. pestis to skin.

The exopolyphosphatase (Ppx) of Pseudomonas aeruginosa is encoded by the PA5241 gene (ppx). Ppx catalyses the hydrolysis of inorganic polyphosphates to orthophosphate (Pi). In the present work, we identified and characterized the promoter region of ppx and its regulation under environmental stress conditions. The role of Ppx in the production of several virulence factors was demonstrated through studies performed on a ppx null mutant. We found that ppx is under the control of two interspaced promoters, dually regulated by nitrogen and phosphate limitation. Under nitrogen-limiting conditions, its expression was controlled from a σ54-dependent promoter activated by the response regulator NtrC. However, under Pi limitation, the expression was controlled from a σ70 promoter, activated by PhoB. Results obtained from the ppx null mutant demonstrated that Ppx is involved in the production of virulence factors associated with both acute infection (e.g. motility-promoting factors, blue/green pigment production, C6–C12 quorum-sensing homoserine lactones) and chronic infection (e.g. rhamnolipids, biofilm formation). Molecular and physiological approaches used in this study indicated that P. aeruginosa maintains consistently proper levels of Ppx regardless of environmental conditions. The precise control of ppx expression appeared to be essential for the survival of P. aeruginosa and the occurrence of either acute or chronic infection in the host.

Leptospirosis is caused by pathogenic species of Leptospira. The aim of this study was to determine and characterize the pathogenicity of four dominant Leptospira isolates prevailing among rats in the Philippines. The isolates were Leptospira interrogans serovar Manilae strain K64, L. interrogans serovar Losbanos strain K37, L. interrogans serovar Ratnapura strain K5 and Leptospira borgpetersenii serovar Javanica strain K6. Pathogenicities were studied using hamsters, which reproduce severe human leptospirosis. The minimum lethal doses were 100 ( = 1) leptospires for K64, K37 and K5, and 101 leptospires for K6. Weight loss amongst the Leptospira-infected hamsters was observed from 1 day before death (K64-, K37- and K5-infected hamsters) to as much as 1 week before death for K6-infected hamsters. Similar and varied gross and microscopic lesions were observed amongst infected hamsters, even for strains belonging to the same species (i.e. L. interrogans). The most significant and common histopathological findings were congestion of the glomerulus, disarrangement of hepatic cords and erythrophagocytosis. Other findings were foamy splenic macrophages for K6, severe petechial pulmonary haemorrhage for K64, and hematuria and severe pulmonary congestion for K37. Immunostaining and culture revealed the presence of leptospires in different organs of the infected hamsters. Based on these results, Leptospira isolates from rats in the Philippines were shown to be highly virulent, causing pulmonary haemorrhage, severe hepato-renal damage and death in hamsters even at lower doses. The present findings on experimental leptospirosis support clinical data showing that patients with severe manifestations of leptospirosis, such as pulmonary haemorrhage, are increasing in the Philippines. These findings may serve as a basis to strengthen the early diagnosis and treatment of human leptospirosis.

Most healthy adults are protected from meningococcal disease by the presence of naturally acquired anti-meningococcal antibodies; however, the identity of the target antigens of this protective immunity remains unclear, particularly for protection against serogroup B disease. To identify the protein targets of natural protective immunity we developed an immunoprecipitation and proteomics approach to define the immunoproteome of the meningococcus. Sera from 10 healthy individuals showing serum bactericidal activity against both a meningococcal C strain (L91543) and the B strain MC58, together with commercially available pooled human sera, were used as probe antisera. Immunoprecipitation was performed with each serum sample and live cells from both meningococcal strains. Immunoprecipitated proteins were identified by MS. Analysis of the immunoproteome from each serum demonstrated both pan-reactive antigens that were recognized by most sera as well as subject-specific antigens. Most antigens were found in both meningococcal strains, but a few were strain-specific. Many of the immunoprecipitated proteins have been characterized previously as surface antigens, including adhesins and proteases, several of which have been recognized as vaccine candidate antigens, e.g. factor H-binding protein, NadA and neisserial heparin-binding antigen. The data demonstrate clearly the presence of meningococcal antibodies in healthy individuals with no history of meningococcal infection and a wide diversity of immune responses. The identification of the immunoreactive proteins of the meningococcus provides a basis for understanding the role of each antigen in the natural immunity associated with carriage and may help to design vaccination strategies.

Thuricin CD is a two component narrow spectrum bacteriocin comprising two peptides with targeted activity against Clostridium difficile. This study examined the bioavailability of thuricin with a view to developing it as an effective antimicrobial against intestinal infection. One of the peptides, Trn-β, was found to be degraded by the gastric enzymes pepsin and α-chymotrypsin both in vitro and in vivo, whereas Trn-α was resistant to digestion by these enzymes and hence was detected in the intestinal porcine digesta following oral ingestion by pigs. In order to determine if spores of the producing organism Bacillus thuringiensis DPC 6431 could be used to deliver the bacteriocin to the gut, spores were fed to 30 mice (approx. 108–2×108 per animal) and their germination, growth and production of thuricin in the gastrointestinal tract (GIT) of the animals was monitored. Almost 99 % of the spores delivered to the GIT were excreted in the first 24 h and neither Trn-α nor Trn-β was detected in the gut or faecal samples of the test mice, indicating that ingestion of B. thuringiensis spores may not be a suitable vehicle for the delivery of thuricin CD. When thuricin CD was delivered rectally to mice (n = 40) and C. difficile shedding monitored at 1, 6, 12 and 24 h post-treatment, there was a >95 % (>1.5 log units) reduction of C. difficile 027 in the colon contents of infected mice (n = 10) 1 h post-treatment compared with the control group (n = 10; P<0.001). Furthermore, 6 h post-treatment there was a further 1.5 log reduction in C. difficile numbers (n = 10) relative to the control group (n = 10; P<0.05). These results would suggest that rectal administration of thuricin may be a promising mode of delivery of thuricin CD to the colon.

Francisella tularensis is a highly infectious Gram-negative pathogen that replicates intracellularly within the mammalian host. One of the factors associated with virulence of F. tularensis is the protein FupA that mediates high-affinity transport of ferrous iron across the outer membrane. Together with its paralogue FslE, a siderophore–ferric iron transporter, FupA supports survival of the pathogen in the host by providing access to the essential nutrient iron. The FupA orthologue in the attenuated live vaccine strain (LVS) is encoded by the hybrid gene fupA/B, the product of an intergenic recombination event that significantly contributes to attenuation of the strain. We used 55Fe transport assays with mutant strains complemented with the different paralogues to show that the FupA/B protein of LVS retains the capacity for high-affinity transport of ferrous iron, albeit less efficiently than FupA of virulent strain Schu S4. 55Fe transport assays using purified siderophore and siderophore-dependent growth assays on iron-limiting agar confirmed previous findings that FupA/B also contributes to siderophore-mediated ferric iron uptake. These assays further demonstrated that the LVS FslE protein is a weaker siderophore–ferric iron transporter than the orthologue from Schu S4, and may be a result of the sequence variation between the two proteins. Our results indicate that iron-uptake mechanisms in LVS differ from those in Schu S4 and that functional differences in the outer membrane iron transporters have distinct effects on growth under iron limitation.