Putative Cluster Research Articles

PyClone: statistical inference of clonal population structure in cancer.

We introduce PyClone, a statistical model for inference of clonal population structures in cancers. PyClone is a Bayesian clustering method for grouping sets of deeply sequenced somatic mutations into putative clonal clusters while estimating their cellular prevalences and accounting for allelic imbalances introduced by segmental copy-number changes and normal-cell contamination. Single-cell sequencing validation demonstrates PyClone's accuracy.

Novel LanT Associated Lantibiotic Clusters Identified by Genome Database Mining

BackgroundFrequent use of antibiotics has led to the emergence of antibiotic resistance in bacteria. Lantibiotic compounds are ribosomally synthesized antimicrobial peptides against which bacteria are not able to produce resistance, hence making them a good alternative to antibiotics. Nisin is the oldest and the most widely used lantibiotic, in food preservation, without having developed any significant resistance against it. Having their antimicrobial potential and a limited number, there is a need to identify novel lantibiotics.Methodology/FindingsIdentification of novel lantibiotic biosynthetic clusters from an ever increasing database of bacterial genomes, can provide a major lead in this direction. In order to achieve this, a strategy was adopted to identify novel lantibiotic biosynthetic clusters by screening the sequenced genomes for LanT homolog, which is a conserved lantibiotic transporter specific to type IB clusters. This strategy resulted in identification of 54 bacterial strains containing the LanT homologs, which are not the known lantibiotic producers. Of these, 24 strains were subjected to a detailed bioinformatic analysis to identify genes encoding for precursor peptides, modification enzyme, immunity and quorum sensing proteins. Eight clusters having two LanM determinants, similar to haloduracin and lichenicidin were identified, along with 13 clusters having a single LanM determinant as in mersacidin biosynthetic cluster. Besides these, orphan LanT homologs were also identified which might be associated with novel bacteriocins, encoded somewhere else in the genome. Three identified gene clusters had a C39 domain containing LanT transporter, associated with the LanBC proteins and double glycine type precursor peptides, the only known example of such a cluster is that of salivaricin.ConclusionThis study led to the identification of 8 novel putative two-component lantibiotic clusters along with 13 having a single LanM and 3 with LanBC genes. Putative lantibiotic clusters identified here hold the potential for the discovery of novel lantibiotic(s).

A Type VI Secretion System Is Involved in Pseudomonas fluorescens Bacterial Competition

Protein secretion systems are crucial mediators of bacterial interactions with other organisms. Among them, the type VI secretion system (T6SS) is widespread in Gram-negative bacteria and appears to inject toxins into competitor bacteria and/or eukaryotic cells. Major human pathogens, such as Vibrio cholerae, Burkholderia and Pseudomonas aeruginosa, express T6SSs. Bacteria prevent self-intoxication by their own T6SS toxins by producing immunity proteins, which interact with the cognate toxins. We describe here an environmental P. fluorescens strain, MFE01, displaying an uncommon oversecretion of Hcp (hemolysin-coregulated protein) and VgrG (valine-glycine repeat protein G) into the culture medium. These proteins are characteristic components of a functional T6SS. The aim of this study was to attribute a role to this energy-consuming overexpression of the T6SS. The genome of MFE01 contains at least two hcp genes (hcp1 and hcp2), suggesting that there may be two putative T6SS clusters. Phenotypic studies have shown that MFE01 is avirulent against various eukaryotic cell models (amebas, plant or animal cell models), but has antibacterial activity against a wide range of competitor bacteria, including rhizobacteria and clinical bacteria. Depending on the prey cell, mutagenesis of the hcp2 gene in MFE01 abolishes or reduces this antibacterial killing activity. Moreover, the introduction of T6SS immunity proteins from S. marcescens, which is not killed by MFE01, protects E. coli against MFE01 killing. These findings suggest that the protein encoded by hcp2 is involved in the killing activity of MFE01 mediated by effectors of the T6SS targeting the peptidoglycan of Gram-negative bacteria. Our results indicate that MFE01 can protect potato tubers against Pectobacterium atrosepticum, which causes tuber soft rot. Pseudomonas fluorescens is often described as a major PGPR (plant growth-promoting rhizobacterium), and our results suggest that there may be a connection between the T6SS and the PGPR properties of this bacterium.

Mass-spectrometry-based spatial proteomics data analysis using pRoloc and pRolocdata

Motivation: Experimental spatial proteomics, i.e. the high-throughput assignment of proteins to sub-cellular compartments based on quantitative proteomics data, promises to shed new light on many biological processes given adequate computational tools.Results: Here we present pRoloc, a complete infrastructure to support and guide the sound analysis of quantitative mass-spectrometry-based spatial proteomics data. It provides functionality for unsupervised and supervised machine learning for data exploration and protein classification and novelty detection to identify new putative sub-cellular clusters. The software builds upon existing infrastructure for data management and data processing.Availability: pRoloc is implemented in the R language and available under an open-source license from the Bioconductor project (http://www.bioconductor.org/). A vignette with a complete tutorial describing data import/export and analysis is included in the package. Test data is available in the companion package pRolocdata.Contact: lg390@cam.ac.uk

Draft Genome Sequence of Streptomyces roseochromogenes subsp. oscitans DS 12.976, Producer of the Aminocoumarin Antibiotic Clorobiocin

Streptomyces roseochromogenes subsp. oscitans DS 12.976 is the producer of the gyrase-inhibiting aminocoumarin antibiotic clorobiocin. Here, we present a draft genome sequence of this strain, in which we identified the clorobiocin gene cluster as well as an unusually high number (43) of further putative secondary metabolite clusters.

Linking the transcriptome and proteome to characterize the venom of the eastern diamondback rattlesnake (Crotalus adamanteus)

Understanding the molecular basis of the phenotype is key to understanding adaptation, and the relationship between genes and specific traits is represented by the genotype-phenotype map. The specialization of the venom-gland towards toxin production enables the use of transcriptomics to identify a large number of loci that contribute to a complex phenotype (i.e., venom), while proteomic techniques allow verification of the secretion of the proteins produced by these loci, creating a genotype-phenotype map. We used the extensive database of mRNA transcripts generated by the venom-gland transcriptome of Crotalus adamanteus along with proteomic techniques to complete the genotype-phenotype map for the C. adamanteus venom system. Nanospray LC/MS(E) analysis of a whole venom sample identified evidence for 52 of the 78 unique putative toxin transcript clusters, including 44 of the 50 most highly expressed transcripts. Tandem mass spectrometry and SDS-PAGE of reversed-phase high-performance liquid chromatography fractions identified 40 toxins which clustered into 20 groups and represented 10 toxin families, creating a genotype-phenotype map. By using the transcriptome to understand the proteome we were able to achieve locus-specific resolution and provide a detailed characterization of the C. adamanteus venom system. Identifying the mechanisms by which genetic variation presents itself to the sieve of selection at the phenotypic level is key to understanding the molecular basis of adaptation, and the first step in understanding this relationship is to identify the genetic basis of the phenotype through the construction of a genotype-phenotype map. We used the high-throughput venom-gland transcriptomic characterization of the eastern diamondback rattlesnake (C. adamanteus) and proteomic techniques to complete and confirm the genotype-phenotype map, providing a detailed characterization of the C. adamanteus venom system.

Genes for iron–sulphur cluster assembly are targets of abiotic stress in rice, Oryza sativa

Iron-sulphur (Fe-S) cluster assembly occurs in chloroplasts, mitochondria and cytosol, involving dozens of genes in higher plants. In this study, we have identified 41 putative Fe-S cluster assembly genes in rice (Oryza sativa) genome, and the expression of all genes was verified. To investigate the role of Fe-S cluster assembly as a metabolic pathway, we applied abiotic stresses to rice seedlings and analysed Fe-S cluster assembly gene expression by qRT-PCR. Our data showed that genes for Fe-S cluster assembly in chloroplasts of leaves are particularly sensitive to heavy metal treatments, and that Fe-S cluster assembly genes in roots were up-regulated in response to iron toxicity, oxidative stress and some heavy metal assault. The effect of each stress treatment on the Fe-S cluster assembly machinery demonstrated an unexpected tissue or organelle specificity, suggesting that the physiological relevance of the Fe-S cluster assembly is more complex than thought. Furthermore, our results may reveal potential candidate genes for molecular breeding of rice.

Genetic structure of Plasmodium falciparum populations across the Honduras-Nicaragua border

BackgroundThe Caribbean coast of Central America remains an area of malaria transmission caused by Plasmodium falciparum despite the fact that morbidity has been reduced in recent years. Parasite populations in that region show interesting characteristics such as chloroquine susceptibility and low mortality rates. Genetic structure and diversity of P. falciparum populations in the Honduras-Nicaragua border were analysed in this study.MethodsSeven neutral microsatellite loci were analysed in 110 P. falciparum isolates from endemic areas of Honduras (n = 77) and Nicaragua (n = 33), mostly from the border region called the Moskitia. Several analyses concerning the genetic diversity, linkage disequilibrium, population structure, molecular variance, and haplotype clustering were conducted.ResultsThere was a low level of genetic diversity in P. falciparum populations from Honduras and Nicaragua. Expected heterozigosity (He) results were similarly low for both populations. A moderate differentiation was revealed by the FST index between both populations, and two putative clusters were defined through a structure analysis. The main cluster grouped most of samples from Honduras and Nicaragua, while the second cluster was smaller and included all the samples from the Siuna community in Nicaragua. This result could partially explain the stronger linkage disequilibrium (LD) in the parasite population from that country. These findings are congruent with the decreasing rates of malaria endemicity in Central America.

The Suf Iron-Sulfur Cluster Synthesis Pathway Is Required for Apicoplast Maintenance in Malaria Parasites

The apicoplast organelle of the malaria parasite Plasmodium falciparum contains metabolic pathways critical for liver-stage and blood-stage development. During the blood stages, parasites lacking an apicoplast can grow in the presence of isopentenyl pyrophosphate (IPP), demonstrating that isoprenoids are the only metabolites produced in the apicoplast which are needed outside of the organelle. Two of the isoprenoid biosynthesis enzymes are predicted to rely on iron-sulfur (FeS) cluster cofactors, however, little is known about FeS cluster synthesis in the parasite or the roles that FeS cluster proteins play in parasite biology. We investigated two putative FeS cluster synthesis pathways (Isc and Suf) focusing on the initial step of sulfur acquisition. In other eukaryotes, these proteins can be located in multiple subcellular compartments, raising the possibility of cross-talk between the pathways or redundant functions. In P. falciparum, SufS and its partner SufE were found exclusively the apicoplast and SufS was shown to have cysteine desulfurase activity in a complementation assay. IscS and its effector Isd11 were solely mitochondrial, suggesting that the Isc pathway cannot contribute to apicoplast FeS cluster synthesis. The Suf pathway was disrupted with a dominant negative mutant resulting in parasites that were only viable when supplemented with IPP. These parasites lacked the apicoplast organelle and its organellar genome – a phenotype not observed when isoprenoid biosynthesis was specifically inhibited with fosmidomycin. Taken together, these results demonstrate that the Suf pathway is essential for parasite survival and has a fundamental role in maintaining the apicoplast organelle in addition to any role in isoprenoid biosynthesis.

Antidepressant effects after short-term and chronic stimulation of the subgenual cingulate gyrus in treatment-resistant depression

BackgroundDeep brain stimulation (DBS) of the subcallosal cingulate gyrus (SCG) is an experimental approach in treatment-resistant depression (TRD). Apart from its potential long-term antidepressant effects acute stimulation effects have been described. We investigated putative neuroanatomical clusters in which such acute effects accumulate and followed patients over the long-term. MethodsWe assessed safety and efficacy of DBS in six patients with TRD receiving bilateral DBS with electrodes implanted in the SCG. First, high intensity 130Hz stimulation was applied on five consecutive days after surgery for 24h comprising a sham condition in a double-blind, randomized design. Acute stimulation was conducted at all four homologous electrode contacts on both sides. Afterwards, chronic stimulation was initiated and the clinical effect was evaluated at 24–36weeks compared to baseline (50% reduction in HAMD scores). The primary outcome criterion was depression severity as assessed with the Hamilton Depression Rating Scale (HAMD-24); secondary outcome parameters were the Montgomery–Åsberg Rating-Scale and Beck Depression Inventory following DBS. The clinical effect over the three scores was compared to sham stimulation and was correlated to the anatomical localization of active contacts by stereotactically delimiting the cluster of most effective contacts in responding patients. ResultsAcute 24h of stimulation showed only moderate reductions in mean HAMD-24, MADRS and BDI scores. At the last observation (24–36weeks), two patients were remitters (HAMD-24<10) and the four other patients were non-responders. ConclusionsOur results confirm that stimulation of the SCG is capable of exerting moderate acute and chronic antidepressant effects. The predictive value of these findings needs to be addressed in future studies.

The sodium/iodide symporter NIS is a transcriptional target of the p53-family members in liver cancer cells

Thyroid iodide accumulation via the sodium/iodide symporter (NIS; SLC5A5) has been the basis for the longtime use of radio-iodide in the diagnosis and treatment of thyroid cancers. NIS is also expressed, but poorly functional, in some non-thyroid human cancers. In particular, it is much more strongly expressed in cholangiocarcinoma (CCA) and hepatocellular carcinoma (HCC) cell lines than in primary human hepatocytes (PHH). The transcription factors and signaling pathways that control NIS overexpression in these cancers is largely unknown. We identified two putative regulatory clusters of p53-responsive elements (p53REs) in the NIS core promoter, and investigated the regulation of NIS transcription by p53-family members in liver cancer cells. NIS promoter activity and endogenous NIS mRNA expression are stimulated by exogenously expressed p53-family members and significantly reduced by member-specific siRNAs. Chromatin immunoprecipitation analysis shows that the p53–REs clusters in the NIS promoter are differentially occupied by the p53-family members to regulate basal and DNA damage-induced NIS transcription. Doxorubicin strongly induces p53 and p73 binding to the NIS promoter, leading to an increased expression of endogenous NIS mRNA and protein in HCC and CCA cells, but not in PHH. Silencing NIS expression reduced doxorubicin-induced apoptosis in HCC cells, pointing to a possible role of a p53-family-dependent expression of NIS in apoptotic cell death. Altogether, these results indicate that the NIS gene is a direct target of the p53 family and suggests that the modulation of NIS by DNA-damaging agents is potentially exploitable to boost NIS upregulation in vivo.

A Tribute to Disorder in the Genome of the Bloom-Forming Freshwater Cyanobacterium Microcystis aeruginosa

Microcystis aeruginosa is one of the most common bloom-forming cyanobacteria in freshwater ecosystems worldwide. This species produces numerous secondary metabolites, including microcystins, which are harmful to human health. We sequenced the genomes of ten strains of M. aeruginosa in order to explore the genomic basis of their ability to occupy varied environments and proliferate. Our findings show that M. aeruginosa genomes are characterized by having a large open pangenome, and that each genome contains similar proportions of core and flexible genes. By comparing the GC content of each gene to the mean value of the whole genome, we estimated that in each genome, around 11% of the genes seem to result from recent horizontal gene transfer events. Moreover, several large gene clusters resulting from HGT (up to 19 kb) have been found, illustrating the ability of this species to integrate such large DNA molecules. It appeared also that all M. aeruginosa displays a large genomic plasticity, which is characterized by a high proportion of repeat sequences and by low synteny values between the strains. Finally, we identified 13 secondary metabolite gene clusters, including three new putative clusters. When comparing the genomes of Microcystis and Prochlorococcus, one of the dominant picocyanobacteria living in marine ecosystems, our findings show that they are characterized by having almost opposite evolutionary strategies, both of which have led to ecological success in their respective environments.

Ferredoxin Competes with Bacterial Frataxin in Binding to the Desulfurase IscS*

The bacterial iron-sulfur cluster (isc) operon is an essential machine that is highly conserved from bacteria to primates and responsible for iron-sulfur cluster biogenesis. Among its components are the genes for the desulfurase IscS that provides sulfur for cluster formation, and a specialized ferredoxin (Fdx) whose role is still unknown. Preliminary evidence suggests that IscS and Fdx interact but nothing is known about the binding site and the role of the interaction. Here, we have characterized the interaction using a combination of biophysical tools and mutagenesis. By modeling the Fdx·IscS complex based on experimental restraints we show that Fdx competes for the binding site of CyaY, the bacterial ortholog of frataxin and sits in a cavity close to the enzyme active site. By in vivo mutagenesis in bacteria we prove the importance of the surface of interaction for cluster formation. Our data provide the first structural insights into the role of Fdx in cluster assembly.

Reversion to susceptibility of a carbapenem-resistant clinical isolate of Klebsiella pneumoniae producing KPC-3

We report the case of a kidney-transplant patient, suffering an intra-abdominal abscess at the surgical site caused by a carbapenem-resistant ST258 Klebsiella pneumoniae clone, producing the KPC-3 carbapenemase. Under tigecycline treatment, the patient developed a sepsis caused by a carbapenem-susceptible ST258 K. pneumoniae strain. Complete DNA sequences of the plasmids carried by the resistant and susceptible strains from this patient were determined. The complete DNA sequences of plasmids were obtained by applying the 454 Genome Sequencer FLX-PLUS procedure on a library constructed of total plasmid DNA purified from the carbapenem-resistant and -susceptible strains. In the carbapenem-resistant strain, four plasmids encoding 24 resistance genes, including blaKPC-3, and two putative virulence clusters were detected. In the susceptible strain, large rearrangements occurred in the KPC-carrying plasmid, causing the deletion of the entire Tn4401::blaKPC-3 transposon, with the consequent reversion of the strain to carbapenem susceptibility. The patient was successfully treated with carbapenems and fully recovered. The description of the plasmid content in these two strains gives interesting insights into the plasticity of KPC-carrying plasmids in K. pneumoniae.

Commentary: Statistical assessment of cancer cluster evidence--in search of a middle ground

As Coory and Jordan point out, probabilities linked to cancer cluster reports must take into account the cherry-picking nature of the reported clusters in order to avoid a large number of false-positive results. These authors must be congratulated for bringing this difficult issue to debate and for summarizing several of the challenges facing researchers. In fact, the challenges are so large that the authors conclude that statistical assessments are impossible to interpret and, rather than assisting, they impair decisionmaking. Their final recommendation is to remove the statistical assessment of chance from protocols for investigating clusters in favour of emphasizing exposure assessment. A principal feature of cancer cluster reports in most countries has been the quick dismissal of the large majority of cases after initial contact. Even for the minority in which an investigation is conducted, neither a plausible biological cause nor an environmental exposure is commonly identified. Goodman et al. evaluated more than 400 reports from cancer cluster investigations carried out since 1990 in the USA, evaluating more than 500 cancers of concern. A statistical increase in incidence was confirmed in 72 of these, but only three were linked to hypothetical exposures, and only one identified a clear cause. The main statistical concern of Coory and Jordan is what they call ‘silent multiple comparisons’ when calculating P-values and confidence intervals associated with the significance of the cancer clusters. Their criticism is valid, since most of these statistical summaries are calculated as if the reported cancer cluster were not picked from among numerous other potential clusters. These unaccounted potential clusters compose the silent comparisons. The authors discuss the use of Bonferroni-type corrections to calculate an approximation for the true P-value associated with given evidence. Coory and Jordan mention also the harder problem that a reference population can be defined arbitrarily. Considering an example of a cluster at the city of Brisbane, they mention that a putative cluster could be evaluated with respect to the population of Brisbane, or the state of Queensland, or the entire Australian population. They show that the P-value can be quite different depending on this choice, if using Bonferroni corrections. There are two problems with this argument. The first is that the public health officials in charge of replying to the cancer reports typically have a population to whom they are accountable. This is the natural reference population. The second problem is that, to carry out the authors’ recommendations, at some point some reference population must be selected, and the conclusions will depend on this selection. The common definition for a disease cluster is the occurrence of a greater than expected number of cases of a particular disease within a group of people, a geographical area or a period of time. It will be virtually impossible to start any cluster investigation, including probing for exposure assessment as the authors suggest, if we do not have a measure of how extreme the occurrence is. One common measure, suggested by the authors, is the standardized incidence ratio, SIR1⁄4O/E, where O is the observed number of cases reported for the cluster, and E is the expected number of cases that would have occurred inside the cluster by taking into account age-specific rates of the entire reference population. Therefore, the SIR itself needs a reference population. Its calculation would also produce different results if the Brisbane, Queensland or Australian population is taken as the reference population, although its impact on E is likely to be much smaller than in Bonferroni correction calculations. Public health officials should have this reference population predetermined before any cluster reports are made. Concerning the multiple comparison problem, statisticians have been working hard as this problem appears in many other application areas such as, for Published by Oxford University Press on behalf of the International Epidemiological Association

Commentary: Assessment of chance should be central in investigation of cancer clusters

As a biostatistician who has considerable experience investigating both fixed putative source clusters and general cluster alarms, and developing methodology for these analyses, I feel I should respond to the various points raised by Coory and Jordan. I believe the authors provide a very narrow view of cluster assessment, characterizing it as testing for an excess of incident cases in a given area. A much broader view has developed over the last two decades, as described below.

A method for processing multivariate data in medical studies

Traditional displays of principal component analyses lack readability to discriminate between putative clusters of variables or cases. Here, the author proposes a method that clusterizes and visualizes variables or cases through principal component analyses thus facilitating their analysis. The method displays pre-determined clusters of variables or cases as urchins that each has a soma (the average point) and spines (the individual variables or cases). Through three examples in the field of neuropsychology, the author illustrates how urchins help examine the modularity of cognitive tasks on the one hand and identify groups of healthy versus brain-damaged participants on the other hand. Some of the data used in this article were obtained from the Alzheimer's Disease Neuroimaging Initiative database. The urchin method was implemented in MATLAB, and the source code is available in the Supporting information. Urchins can be useful in biomedical studies to identify distinct phenomena at first glance, each having several measures (clusters of variables) or distinct groups of participants (clusters of cases).

Genome Sequence of Lactobacillus pentosus KCA1: Vaginal Isolate from a Healthy Premenopausal Woman

The vaginal microbiota, in particular Lactobacillus species, play an important role in female health through modulation of immunity, countering pathogens and maintaining a pH below 4.7. We report the isolation and genome sequence of Lactobacillus pentosus strain KCA1 (formally known as L. plantarum) from the vagina of a healthy Nigerian woman. The genome was sequenced using Illumina GA II technology. The resulting 16,920,226 paired-end reads were assembled with the Velvet tool. Contigs were annotated using the RAST server, and manually curated. A comparative analysis with the available genomes of L. pentosus IG1 and L. plantarum WCFS1 showed that over 15% of the predicted functional activities are found only in this strain. The strain has a chromosome sequence of 3,418,159 bp with a G+C content of 46.4%, and is devoid of plasmids. Novel gene clusters or variants of known genes relative to the reference genomes were found. In particular, the strain has loci encoding additional putative mannose phosphotransferase systems. Clusters of genes include those for utilization of hydantoin, isopropylmalate, malonate, rhamnosides, and genes for assimilation of polyglycans, suggesting the metabolic versatility of L. pentosus KCA1. Loci encoding putative phage defense systems were also found including clustered regularly interspaced short palindromic repeats (CRISPRs), abortive infection (Abi) systems and toxin-antitoxin systems (TA). A putative cluster of genes for biosynthesis of a cyclic bacteriocin precursor, here designated as pentocin KCA1 (penA) were identified. These findings add crucial information for understanding the genomic and geographic diversity of vaginal lactobacilli.

A comparative hidden Markov model analysis pipeline identifies proteins characteristic of cereal-infecting fungi

BackgroundFungal pathogens cause devastating losses in economically important cereal crops by utilising pathogen proteins to infect host plants. Secreted pathogen proteins are referred to as effectors and have thus far been identified by selecting small, cysteine-rich peptides from the secretome despite increasing evidence that not all effectors share these attributes.ResultsWe take advantage of the availability of sequenced fungal genomes and present an unbiased method for finding putative pathogen proteins and secreted effectors in a query genome via comparative hidden Markov model analyses followed by unsupervised protein clustering. Our method returns experimentally validated fungal effectors in Stagonospora nodorum and Fusarium oxysporum as well as the N-terminal Y/F/WxC-motif from the barley powdery mildew pathogen. Application to the cereal pathogen Fusarium graminearum reveals a secreted phosphorylcholine phosphatase that is characteristic of hemibiotrophic and necrotrophic cereal pathogens and shares an ancient selection process with bacterial plant pathogens. Three F. graminearum protein clusters are found with an enriched secretion signal. One of these putative effector clusters contains proteins that share a [SG]-P-C-[KR]-P sequence motif in the N-terminal and show features not commonly associated with fungal effectors. This motif is conserved in secreted pathogenic Fusarium proteins and a prime candidate for functional testing.ConclusionsOur pipeline has successfully uncovered conservation patterns, putative effectors and motifs of fungal pathogens that would have been overlooked by existing approaches that identify effectors as small, secreted, cysteine-rich peptides. It can be applied to any pathogenic proteome data, such as microbial pathogen data of plants and other organisms.

Pilus Biogenesis in Lactococcus lactis: Molecular Characterization and Role in Aggregation and Biofilm Formation

The genome of Lactococcus lactis strain IL1403 harbors a putative pilus biogenesis cluster consisting of a sortase C gene flanked by 3 LPxTG protein encoding genes (yhgD, yhgE, and yhhB), called here pil. However, pili were not detected under standard growth conditions. Over-expression of the pil operon resulted in production and display of pili on the surface of lactococci. Functional analysis of the pilus biogenesis machinery indicated that the pilus shaft is formed by oligomers of the YhgE pilin, that the pilus cap is formed by the YhgD pilin and that YhhB is the basal pilin allowing the tethering of the pilus fibers to the cell wall. Oligomerization of pilin subunits was catalyzed by sortase C while anchoring of pili to the cell wall was mediated by sortase A. Piliated L. lactis cells exhibited an auto-aggregation phenotype in liquid cultures, which was attributed to the polymerization of major pilin, YhgE. The piliated lactococci formed thicker, more aerial biofilms compared to those produced by non-piliated bacteria. This phenotype was attributed to oligomers of YhgE. This study provides the first dissection of the pilus biogenesis machinery in a non-pathogenic Gram-positive bacterium. Analysis of natural lactococci isolates from clinical and vegetal environments showed pili production under standard growth conditions. The identification of functional pili in lactococci suggests that the changes they promote in aggregation and biofilm formation may be important for the natural lifestyle as well as for applications in which these bacteria are used.