- Volume 159, Issue Pt_6, 2013

Difficulty overexpressing (eukaryotic) membrane proteins is generally considered as the major impediment in their structural and functional research. Lactococcus lactis possesses many properties ideal for membrane protein expression. In order to investigate membrane protein expression in L. lactis, we created a novel expression system by introducing Mistic, a short peptide previously identified in Bacillus subtilis, into L. lactis. The potential of this system was demonstrated in the overexpression of a eukaryotic membrane protein (pkjDes4) and a prokaryotic membrane protein (pkjLi), a newly isolated linoleate isomerase from Lactobacillus acidophilus. The expression levels reached up to 4.4 % and 45.2 % for pkjDes4 and pkjLi, respectively, which represented an exceptionally robust ability to overproduce membrane proteins. Moreover, the expressed pkjLi was functional, with its catalysing nature characterized for the first time in this species. Up to 0.852 mg ml−1 conjugated linoleic acid was obtained during the linoleic acid conversion catalysed by the recombinant lactococcal strains. In summary, we established a membrane protein expression system in L. lactis and examined its functionality. Our results demonstrate that the Mistic chaperoning strategy can be efficiently applied to L. lactis hosts and show its extraordinary capacity to facilitate the high-yield production of intractable membrane proteins.

Undisturbed plasmid dynamics is required for the stable maintenance of plasmid DNA in bacterial cells. In this work, we analysed subcellular localization, DNA synthesis and nucleoprotein complex formation of plasmid RK2 during the cell cycle of Caulobacter crescentus. Our microscopic observations showed asymmetrical distribution of plasmid RK2 foci between the two compartments of Caulobacter predivisional cells, resulting in asymmetrical allocation of plasmids to progeny cells. Moreover, using a quantitative PCR (qPCR) method, we estimated that multiple plasmid particles form a single fluorescent focus and that the number of plasmids per focus is approximately equal in both swarmer and predivisional Caulobacter cells. Analysis of the dynamics of TrfA–oriV complex formation during the Caulobacter cell cycle revealed that TrfA binds oriV primarily during the G1 phase, however, plasmid DNA synthesis occurs during the S and G2 phases of the Caulobacter cell cycle. Both in vitro and in vivo analysis of RK2 replication initiation in C. crescentus cells demonstrated that it is independent of the Caulobacter DnaA protein in the presence of the longer version of TrfA protein, TrfA-44. However, in vivo stability tests of plasmid RK2 derivatives suggested that a DnaA-dependent mode of plasmid replication initiation is also possible.

ϕEf11 is a temperate bacteriophage originally isolated by induction from a lysogenic Enterococcus faecalis strain recovered from an infected root canal, and the ϕEf11 prophage is widely disseminated among strains of E. faecalis. Because E. faecalis has emerged as a significant opportunistic human pathogen, we were interested in examining the genes and regulatory sequences predicted to be critical in the establishment/maintenance of lysogeny by ϕEf11 as a first step in the construction of the genome of a virulent, highly lytic phage that could be used in treating serious E. faecalis infections. Passage of ϕEf11 in E. faecalis JH2-2 yielded a variant that produced large, extensively spreading plaques in lawns of indicator cells, and elevated phage titres in broth cultures. Genetic analysis of the cloned virus producing the large plaques revealed that the variant was a recombinant between ϕEf11 and a defective ϕFL1C-like prophage located in the E. faecalis JH2-2 chromosome. The recombinant possessed five ORFs of the defective ϕFL1C-like prophage in place of six ORFs of the ϕEf11 genome. Deletion of the putative lysogeny gene module (ORFs 31–36) and replacement of the putative cro promoter from the recombinant phage genome with a nisin-inducible promoter resulted in no loss of virus infectivity. The genetic construct incorporating all the aforementioned ϕEf11 genomic modifications resulted in the generation of a variant that was incapable of lysogeny and insensitive to repressor, rendering it virulent and highly lytic, with a notably extended host range.

The O-polysaccharide was isolated by mild acid hydrolysis of the lipopolysaccharide of Proteus vulgaris HSC 438, and the following structure was established by chemical methods and one- and two-dimensional 1H and 13C NMR spectroscopy: →3)-β-d-Quip4NAlo-(1→3)-α-d-Galp6Ac-(1→6)-α-d-Glcp-(1→3)-α-l-FucpNAc-(1→3)-β-d-GlcpNAc-(1→, where d-Qui4N stands for 4-amino-4,6-dideoxy-d-glucose and Alo for N-((S)-1-carboxyethyl)-l-alanine (alanopine); only about half of the Gal residues are O-acetylated. This structure is unique among the Proteus O-polysaccharides, and therefore it is proposed to classify P. vulgaris HSC 438 into a new Proteus serogroup, O76. A serological cross-reactivity of HSC 438 O-antiserum and lipopolysaccharides of some other Proteus serogroups was observed and accounted for by shared epitopes on the O-polysaccharides or lipopolysaccharide core regions, including that associated with d-Qui4NAlo.

Iron is an essential element required for most organisms. The high-affinity iron-uptake systems in the opportunistic pathogen Candida albicans are activated under iron-limited conditions and are also required for virulence. Here one component of high-affinity iron-uptake systems, the multicopper oxidase (MCO) genes, was characterized. We examined the expression of five MCO genes and demonstrated that CaFET3 and CaFET34 were the major MCO genes in response to iron deficiency. Complementation of the Saccharomyces cerevisiae fet3Δ mutant showed that CaFET34 could effectively rescue the growth phenotype in iron-limited medium. Deletion of CaFET33 and CaFET34 in C. albicans decreased cellular iron content and iron acquisition during iron starvation. However, the fet33Δ/Δ and fet34Δ/Δ mutants exhibited no obvious growth defect in solid iron-limited medium while the fet34Δ/Δ mutant showed a slight growth defect in liquid medium. Further analysis shows that other members of the five MCO genes, especially CaFET3, would compensate for the absence of CaFET33 and CaFET34. Furthermore, for the first time, we provide evidence that CaFET34 is implicated in hyphal development in an iron-independent manner and is required for C. albicans virulence in a mouse model of systemic infection. Together, our results not only expand our understanding about the expression of the MCO genes in C. albicans, but also provide a novel insight into the role of CaFET34 in iron metabolism, hyphal development and virulence.

Foodborne illness due to bacterial pathogens is increasing worldwide as a consequence of the higher consumption of fresh and minimally processed food products, which are more easily cross-contaminated. The efficiency of food pasteurization methods is usually measured by c.f.u. plate counts, a method discriminating viable from dead cells on the basis of the ability of cells to replicate and form colonies on standard growth media, thus ignoring viable but not cultivable cells. Supercritical CO2 (SC-CO2) has recently emerged as one of the most promising fresh food pasteurization techniques, as an alternative to traditional, heat-based methods. In the present work, using three SC-CO2-treated foodborne bacteria (Listeria monocytogenes, Salmonella enterica and Escherichia coli) we tested and compared the performance of alternative viability test methods based on membrane permeability: propidium monoazide quantitative PCR (PMA-qPCR) and flow cytometry (FCM). Results were compared based on plate counts and fluorescent microscopy measurements, which showed that the former dramatically reduced the number of cultivable cells by more than 5 log units. Conversely, FCM provided a much more detailed picture of the process, as it directly quantifies the number of total cells and distinguishes among three categories, including intact, partially permeabilized and permeabilized cells. A comparison of both PMA-qPCR and FCM with plate count data indicated that only a fraction of intact cells maintained the ability to replicate in vitro. Following SC-CO2 treatment, FCM analysis revealed a markedly higher level of bacterial membrane permeabilization of L. monocytogenes with respect to E. coli and S. enterica. Furthermore, an intermediate permeabilization state in which the cellular surface was altered and biovolume increased up to 1.5-fold was observed in L. monocytogenes, but not in E. coli or S. enterica. FCM thus compared favourably with other methods and should be considered as an accurate analytical tool for applications in which monitoring bacterial viability status is of importance, such as microbiological risk assessment in the food chain or in the environment.

The general stress response of alphaproteobacteria is regulated by a partner-switching mechanism that involves the alternative sigma factor σEcfG, the anti-sigma factor NepR and the anti-sigma factor antagonist PhyR. To address the question of how the PhyR–NepR–σEcfG cascade is activated and modulated in Methylobacterium extorquens, a forward genetic screen was applied. The screen identified the single-domain response regulator Mext_0407 as a novel regulatory element in the general stress response of M. extorquens. Analysis of phenotypes and of transcriptional fusions of PhyR-dependent genes shows that the mext_0407 deletion mutant fails to respond to various stresses. Mext_0407 requires the putative phosphorylatable aspartate-64 for its activity in vivo and genetic evidence indicates that Mext_0407 operates upstream of the PhyR–NepR–σEcfG cascade.

The ascomycete fungus Fusarium graminearum is a devastating plant pathogen for major cereal crops. Ascospores are produced via sexual reproduction and forcibly discharged from mature perithecia, which function as the primary inocula. Perithecium development involves complex cellular processes and is under polygenic control. In this study, a novel gene, GEA1, was found to be required for ascus wall development in F. graminearum. GEA1 deletion mutants produced normal-shaped perithecia and ascospores, yet ascospores were observed to precociously germinate inside the perithecium. Moreover, GEA1 deletions resulted in abnormal ascus walls that collapsed prior to ascospore discharge. Based on localization of GEA1 to plasma membrane, GEA1 may be directly involved in ascus wall biogenesis. This is the first report to identify a unique gene required for ascus wall development in F. graminearum.

Previous studies have demonstrated that Pseudomonas putida strains are not only capable of growth on a wide range of organic substrates, but also chemotactic towards many of these compounds. However, in most cases the specific chemoreceptors that are involved have not been identified. The complete genome sequences of P. putida strains F1 and KT2440 revealed that each strain is predicted to encode 27 methyl-accepting chemotaxis proteins (MCPs) or MCP-like proteins, 25 of which are shared by both strains. It was expected that orthologous MCPs in closely related strains of the same species would be functionally equivalent. However, deletion of the gene encoding the P. putida F1 orthologue (locus tag Pput_4520, designated mcfS) of McpS, a known receptor for organic acids in P. putida KT2440, did not result in an obvious chemotaxis phenotype. Therefore, we constructed individual markerless MCP gene deletion mutants in P. putida F1 and screened for defective sensory responses to succinate, malate, fumarate and citrate. This screen resulted in the identification of a receptor, McfQ (locus tag Pput_4894), which responds to citrate and fumarate. An additional receptor, McfR (locus tag Pput_0339), which detects succinate, malate and fumarate, was found by individually expressing each of the 18 genes encoding canonical MCPs from strain F1 in a KT2440 mcpS-deletion mutant. Expression of mcfS in the same mcpS deletion mutant demonstrated that, like McfR, McfS responds to succinate, malate, citrate and fumarate. Therefore, at least three receptors, McfR, McfS, and McfQ, work in concert to detect organic acids in P. putida F1.

σB is an alternative σ factor that regulates stress response and virulence genes in the foodborne pathogen Listeria monocytogenes. To gain further insight into σB-dependent regulatory mechanisms in L. monocytogenes, we (i) performed quantitative proteomic comparisons between the L. monocytogenes parent strain 10403S and an isogenic ΔsigB mutant and (ii) conducted a meta-analysis of published microarray studies on the 10403S σB regulon. A total of 134 genes were found to be significantly positively regulated by σB at the transcriptomic level with >75 % of these genes preceded by putative σB-dependent promoters; 21 of these 134 genes were also found to be positively regulated by σB through proteomics. In addition, 15 proteins were only found to be positively regulated by σB through proteomics analyses, including Lmo1349, a putative glycine cleavage system protein. The lmo1349 gene is preceded by a 5′ UTR that functions as a glycine riboswitch, which suggests regulation of glycine metabolism by σB in L. monocytogenes. Herein, we propose a model where σB upregulates pathways that facilitate biosynthesis and uptake of glycine, which may then activate this riboswitch. Our data also (i) identified a number of σB-dependent proteins that appear to be encoded by genes that are co-regulated by multiple transcriptional regulators, in particular PrfA, and (ii) found σB-dependent genes and proteins to be overrepresented in the ‘energy metabolism’ role category, highlighting contributions of the σB regulon to L. monocytogenes energy metabolism as well as a role of PrfA and σB interaction in regulating aspects of energy metabolism in L. monocytogenes.

Previously, we have identified an avirulent Ralstonia solanacearum mutant carrying a transposon insertion in RSc0411, a gene homologous to the Escherichia coli LPS-transporting protein LptC. However, how the disruption of RSc0411 affects the bacterium–plant interactions and leads to decreased pathogenicity was not known. Here we show that the disruption of RSc0411 leads to pleiotropic defects, including reducing bacterial motility, biofilm formation, root attachment, rough-form LPS production and virulence in tomato and increasing membrane permeability. Disruption of the orthologous RSc0411 present in other R. solanacearum strains proves that most of these functions are conserved in the species. In contrast, trans-complementation analyses show that only RSc0411 orthologues from closely related bacteria can rescue the defects of the disruption mutant. These results enable us to propose a function for RSc0411, and for the clustered genes, in LPS biogenesis, and for the first time, to our knowledge, also a role of a gene from the DUF1239 gene family in bacterial pathogenicity. In addition and notably, the RSc0411 mutant displays a strain-specific phenotype for hypersensitive response (HR), in which the RSc0411 disruption impairs the HR caused by strain Pss190 but not that by strain Pss1308. Consistent with this strain-specific defect, the mutation clearly affects expression of the type III secretion system (T3SS) in Pss190 but not in other strains, suggesting that the HR-deficient phenotype of the RSc0411 mutant in Pss190 is due to impairment of the T3SS and thus RSc0411 has a strain-specific role in the T3SS activity of R. solanacearum.

The genome of Xanthomonas albilineans, the causal agent of sugar cane leaf scald, carries a gene cluster encoding a predicted quorum sensing system that is highly related to the diffusible signalling factor (DSF) systems of the plant pathogens Xylella fastidiosa and Xanthomonas campestris. In these latter pathogens, a cluster of regulation of pathogenicity factors (rpf) genes encodes the DSF system and is involved in control of various cellular processes. Mutation of Xanthomonas albilineans rpfF, encoding a predicted DSF synthase, in Florida strain XaFL07-1 resulted in a small reduction of disease severity (DS). Single-knockout mutations of rpfC and rpfG (encoding a predicted DSF sensor and regulator, respectively) had no effect on DS or swimming motility of the pathogen. However, capacity of the pathogen to cause disease was slightly reduced and swimming motility was severely affected when rpfG and rpfC were both deleted. Similar results were obtained when the entire rpfGCF region was deleted. Surprisingly, when the pathogen was mutated in rpfG or rpfC (single or double mutations) it was able to colonize sugar cane spatially more efficiently than the wild-type. Mutation in rpfF alone did not affect the degree of spatial invasion. We conclude that the DSF signal contributes to symptom expression but not to invasion of sugar cane stalks by Xanthomonas albilineans strain XaFL07-1, which is mainly controlled by the RpfCG two-component system.

The opportunistic fungal pathogen Candida albicans is one of the leading agents of life-threatening infections affecting immunocompromised individuals. Many factors make C. albicans a successful pathogen. These include the ability to switch between yeast and invasive hyphal morphologies in addition to an arsenal of cell wall virulence factors such as lipases, proteases, dismutases and adhesins that promote the attachment to the host, a prerequisite for invasive growth. We have previously characterized Hwp2, a C. albicans cell wall protein which we found necessary for proper oxidative stress, biofilm formation and adhesion to host cells. Baker’s yeast Saccharomyces cerevisiae also possesses adhesins that promote aggregation and flocculence. Flo11 is one such adhesin that has sequence similarity to Hwp2. Here we determined that transforming an HWP2 cassette can complement the lack of filamentation of an S. cerevisiae flo11 null strain and impart on S. cerevisiae adhesive properties similar to those of a pathogen.

Transcriptional regulation mediates adaptation of pathogens to environmental stimuli and is important for host colonization. The Campylobacter jejuni genome sequence reveals a surprisingly small set of regulators, mostly of unknown function, suggesting an intricate regulatory network. Interestingly, C. jejuni lacks the homologues of ubiquitous regulators involved in stress response found in many other Gram-negative bacteria. Nonetheless, cj1000 is predicted to encode the sole LysR-type regulator in the C. jejuni genome, and thus may be involved in major adaptation pathways. A cj1000 mutant strain was constructed and found to be attenuated in its ability to colonize 1-day-old chicks. Complementation of the cj1000 mutation restored the colonization ability to wild-type levels. The mutant strain was also outcompeted in a competitive colonization assay of the piglet intestine. Oxygraphy was carried out for what is believed to be the first time with the Oroboros Oxygraph-2k on C. jejuni and revealed a role for Cj1000 in controlling O2 consumption. Furthermore, microarray analysis of the cj1000 mutant revealed both direct and indirect regulatory targets, including genes involved in energy metabolism and oxidative stress defences. These results highlight the importance of Cj1000 regulation in host colonization and in major physiological pathways.

Lactococcus lactis possesses a pronounced extracellular Cu2+-reduction activity which leads to the accumulation of Cu+ in the medium. The kinetics of this reaction were not saturable by increasing copper concentrations, suggesting a non-enzymic reaction. A copper-reductase-deficient mutant, isolated by random transposon mutagenesis, had an insertion in the menE gene, which encodes O-succinylbenzoic acid CoA ligase. This is a key enzyme in menaquinone biosynthesis. The ΔmenE mutant was deficient in short-chain menaquinones, and exogenously added menaquinone complemented the copper-reductase-deficient phenotype. Haem-induced respiration of wild-type L. lactis efficiently suppressed copper reduction, presumably by competition by the bd-type quinol oxidase for menaquinone. As expected, the ΔmenE mutant was respiration-deficient, but could be made respiration-proficient by supplementation with menaquinone. Growth of wild-type cells was more copper-sensitive than that of the ΔmenE mutant, due to the production of Cu+ ions by the wild-type. This growth inhibition of the wild-type was strongly attenuated if Cu+ was scavenged with the Cu(I) chelator bicinchoninic acid. These findings support a model whereby copper is non-enzymically reduced at the membrane by menaquinones. Respiration effectively competes for reduced quinones, which suppresses copper reduction. These findings highlight novel links between copper reduction, respiration and Cu+ toxicity in L. lactis.

Crenarchaeota include extremely thermoacidophilic organisms that thrive in geothermal environments dominated by sulfidic ores and heavy metals such as mercury. Mercuric ion, Hg(II), inactivates transcription in the crenarchaeote Sulfolobus solfataricus and simultaneously derepresses transcription of a resistance operon, merHAI, through interaction with the MerR transcription factor. While mercuric reductase (MerA) is required for metal resistance, the role of MerH, an adjacent small and predicted product of an ORF, has not been explored. Inactivation of MerH either by nonsense mutation or by in-frame deletion diminished Hg(II) resistance of mutant cells. Promoter mapping studies indicated that Hg(II) sensitivity of the merH nonsense mutant arose through transcriptional polarity, and its metal resistance was restored partially by single copy merH complementation. Since MerH was not required in vitro for MerA-catalysed Hg(II) reduction, MerH may play an alternative role in metal resistance. Inductively coupled plasma-mass spectrometry analysis of the MerH deletion strain following metal challenge indicated that there was prolonged retention of intracellular Hg(II). Finally, a reduced rate of mer operon induction in the merH deletion mutant suggested that the requirement for MerH could result from metal trafficking to the MerR transcription factor.