Capsular Polysaccharide Production Research Articles

The phosphocarrier protein HPr of Neisseria meningitidis interacts with the transcription regulator CrgA and its deletion affects capsule production, cell adhesion, and virulence

The bacterial phosphotransferase system (PTS) transports and phosphorylates sugars, but also carries out numerous regulatory functions. The β-proteobacterium Neisseria meningitidis possesses an incomplete PTS unable to transport carbon sources because it lacks a membrane component. Nevertheless, the residual phosphorylation cascade is functional and the meningococcal PTS was therefore expected to carry out regulatory roles. Interestingly, a ΔptsH mutant (lacks the PTS protein HPr) exhibited reduced virulence in mice and after intraperitoneal challenge it was rapidly cleared from the bloodstream of BALB/c mice. The rapid clearance correlates with lower capsular polysaccharide production by the ΔptsH mutant, which is probably also responsible for its increased adhesion to Hec-1-B epithelial cells. In addition, compared to the wild-type strain more apoptotic cells were detected when Hec-1-B cells were infected with the ΔptsH strain. Coimmunoprecipitation revealed an interaction of HPr and P-Ser-HPr with the LysR type transcription regulator CrgA, which among others controls its own expression. Moreover, ptsH deletion caused increased expression of a ΦcrgA-lacZ fusion. Finally, the presence of HPr or phospho-HPr's during electrophoretic mobility shift assays enhanced the affinity of CrgA for its target sites preceding crgA and pilE, but HPr did not promote CrgA binding to the sia and pilC1 promoter regions.

Investigation of Appropriate Cultivation Approach for Capsular Polysaccharide Production by Streptococcus pneumoniae Serotype 19F

With the aim of determining an appropriate cultivation approach for the capsular polysaccharide production by Streptococcus pneumoniae serotype 19F, the influence of environmental and culture medium conditions on the pneumococcal culture was investigated. Using 5% CO2 atmosphere instead of using an aeration limited environment enhanced the capsule production 3.5 fold. Buffering the cultivation medium prevented the culture pH drop to the acidic condition and increased the capsule production almost 5 fold. Utilizing casamino acid as the nitrogen source of the culture medium instead of soytone provided 1.3 fold increase in the capsule production. Glucose, sorbitol, lactose and sucrose were investigated as carbon sources of the culture medium. Regarding costs of these sugars and their effects on the capsule production, lactose was the best carbon source. Our results demonstrated that buffering the cultivation medium had the most profound effect on the serotype 19F capsule production. The capsule was produced at 1.706 mg/ml in the buffered medium. Applying this culture method allows the cost effective production of the serotype 19F pneumococcal capsule for inclusion in pneumococcal vaccines.

Autophosphorylation of the Bacterial Tyrosine-Kinase CpsD Connects Capsule Synthesis with the Cell Cycle in Streptococcus pneumoniae.

Bacterial capsular polysaccharides (CPS) are produced by a multi-protein membrane complex, in which a particular type of tyrosine-autokinases named BY-kinases, regulate their polymerization and export. However, our understanding of the role of BY-kinases in these processes remains incomplete. In the human pathogen Streptococcus pneumoniae, the BY-kinase CpsD localizes at the division site and participates in the proper assembly of the capsule. In this study, we show that the cytoplasmic C-terminal end of the transmembrane protein CpsC is required for CpsD autophosphorylation and localization at mid-cell. Importantly, we demonstrate that the CpsC/CpsD complex captures the polysaccharide polymerase CpsH at the division site. Together with the finding that capsule is not produced at the division site in cpsD and cpsC mutants, these data show that CPS production occurs exclusively at mid-cell and is tightly dependent on CpsD interaction with CpsC. Next, we have analyzed the impact of CpsD phosphorylation on CPS production. We show that dephosphorylation of CpsD induces defective capsule production at the septum together with aberrant cell elongation and nucleoid defects. We observe that the cell division protein FtsZ assembles and localizes properly although cell constriction is impaired. DAPI staining together with localization of the histone-like protein HlpA further show that chromosome replication and/or segregation is defective suggesting that CpsD autophosphorylation interferes with these processes thus resulting in cell constriction defects and cell elongation. We show that CpsD shares structural homology with ParA-like ATPases and that it interacts with the chromosome partitioning protein ParB. Total internal reflection fluorescence microscopy imaging demonstrates that CpsD phosphorylation modulates the mobility of ParB. These data support a model in which phosphorylation of CpsD acts as a signaling system coordinating CPS synthesis with chromosome segregation to ensure that daughter cells are properly wrapped in CPS.

Capsular polysaccharide production by Streptococcus pneumoniae serotype 1: from strain selection to fed-batch cultivation.

Streptococcus pneumoniae is a human pathogen largely transmitted by aerosols. Vaccines are the main strategy against this pathogen, and the capsular polysaccharide (PS) is its major antigen. S. pneumoniae serotype 1 is associated with large outbreaks and epidemics of invasive diseases. The aims of this work were to screen serotype 1 strains to identify the best PS1 producer, evaluate three peptones for PS1 production, investigate the effects of culture medium components using a design of experiments (DoE), a statistic tool for optimization, and propose a new medium/cultivation strategy. After flask cultivation of nine strains, two that produced high PS1 and biomass values were chosen for further evaluation in the bioreactor, and ST595/01 was chosen as the best PS1 producer strain. Among the peptones tested (Casamino acids, Soytone, and Phytone), the highest PS1 production (298mg/L) was reached with Phytone. Next, DoE (2(4-1)) was performed to evaluate the effects of yeast extract (YE), Phytone, L-asparagine (Asn), and L-glutamine (Gln), yielding the following results: Phytone presented positive effects (p < 0.05) for maximum production of biomass, PS1, acetate, and lactate; YE showed positive effects for biomass and acid production (p < 0.05); Gln exerted a minor positive effect on PS1 yield factor on glucose (p < 0.1); and Asn presented only an effect on acetate production (p < 0.1). Hence, a new culture medium was formulated based on Phytone, YE, and glucose, and batch and fed-batch cultivations were evaluated. The fed-batch cultivation showed almost 2 times the biomass and 2.5 times the PS1 production as the batch culture, and 8-10 times higher PS1 production than has been previously reported.

Loss of hypermucoviscosity and increased fitness cost in colistin-resistant Klebsiella pneumoniae sequence type 23 strains.

In this study, we investigated the effects of colistin resistance on virulence and fitness in hypermucoviscous (HV) Klebsiella pneumoniae sequence type 23 (ST23) strains. Colistin-resistant mutants were developed from three colistin-susceptible HV K. pneumoniae ST23 strains. The lipid A structures of strains were analyzed by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry. Changes in HV were investigated using the string test, and extracellular polysaccharide production was quantified. The expression levels of the phoQ, pmrD, pmrB, pbgP, magA, and p-rmpA2 genes, serum resistance, and biofilm-forming activity were determined. The fitness of colistin-resistant mutants compared to that of the parental strains was examined by determining the competitive index (CI). The colistin-resistant mutants exhibited reduced HV, which was accompanied by decreased formation of capsular polysaccharides (CPS) and reduced expression of genes (magA and p-rmpA2). While there was enhanced expression of pmrD and pbgP in all colistin-resistant derivatives, there were differences in the expression levels of phoQ and pmrB between strains. MALDI-TOF analysis detected the addition of aminoarabinose or palmitate to the lipid A moiety of lipopolysaccharide in the colistin-resistant derivatives. In addition, survival rates in the presence of normal human serum were decreased in the mutant strains, and CI values (0.01 to 0.19) indicated significant fitness defects in the colistin-resistant derivatives compared to the respective parental strains. In hypervirulent HV K. pneumoniae strains, the acquisition of colistin resistance was accompanied by reduced CPS production, impaired virulence, and a significant fitness cost.

Characterization of self-generated variants in Pseudoalteromonas lipolytica biofilm with increased antifouling activities

Pseudoalteromonas is widespread in various marine environments, and most strains can affect invertebrate larval settlement and metamorphosis by forming biofilms. However, the impact and the molecular basis of population diversification occurring in Pseudoalteromonas biofilms are poorly understood. Here, we show that morphological diversification is prevalent in Pseudoalteromonas species during biofilm formation. Two types of genetic variants, wrinkled (frequency of 12 ± 5 %) and translucent (frequency of 5 ± 3 %), were found in Pseudoalteromonas lipolytica biofilms. The inducing activities of biofilms formed by the two variants on larval settlement and metamorphosis of the mussel Mytilus coruscus were significantly decreased, suggesting strong antifouling activities. Using whole-genome re-sequencing combined with genetic manipulation, two genes were identified to be responsible for the morphology alternations. A nonsense mutation in AT00_08765 led to a wrinkled morphology due to the overproduction of cellulose, whereas a point mutation in AT00_17125 led to a translucent morphology via a reduction in capsular polysaccharide production. Taken together, the results suggest that the microbial behavior on larval settlement and metamorphosis in marine environment could be affected by the self-generated variants generated during the formation of marine biofilms, thereby rendering potential application in biocontrol of marine biofouling.Electronic supplementary materialThe online version of this article (doi:10.1007/s00253-015-6865-x) contains supplementary material, which is available to authorized users.

Engineering a branch of the UDP-precursor biosynthesis pathway enhances the production of capsular polysaccharide in Escherichia coli O5:K4:H4.

Escherichia coli K4 produces a capsule with a chemical structure that resembles chondroitin, a molecule with established chondro protective properties. The endogenous genes pgm and galU are involved in the biosynthesis of UDP-glucose which is a critical intermediate in carbohydrate metabolism and biochemical precursor of UDP-glucuronic acid. Together with UDP-N-acetylgalactosamine, UDP-glucuronic acid is used as sugar donor for capsule biosynthesis. The aim of the study was to evaluate how a change in the pathways leading to UDP-glucuronic acid biosynthesis affected capsular polysaccharide production. One additional copy of pgm and galU was introduced in E. coli K4 and in the previously described recombinant strain EcK4r3. A microbioreactor was used to analyse strain performance with parallel batch experiments, demonstrating increased polysaccharide concentrations and providing data that are comparable to those obtained in larger fermenters. Further experiments on a glutamine enriched medium showed an additional 45% increase of capsule production, maybe indicating the need to balance both branches leading to polymer biosynthesis in order to maximize yields. In the effort towards the establishment of a feasible bio-chondroitin production process this study provides information on how the availability of sugar precursors impacts polysaccharide biosynthesis in E. coli K4, a complex unexplored aspect of a multifaceted process.

USA300 and USA500 clonal lineages of Staphylococcus aureus do not produce a capsular polysaccharide due to conserved mutations in the cap5 locus.

ABSTRACTThe surface capsular polysaccharide (CP) is a virulence factor that has been used as an antigen in several successful vaccines against bacterial pathogens. A vaccine has not yet been licensed against Staphylococcus aureus, although two multicomponent vaccines that contain CP antigens are in clinical trials. In this study, we evaluated CP production in USA300 methicillin-resistant S. aureus (MRSA) isolates that have become the predominant community-associated MRSA clones in the United States. We found that all 167 USA300 MRSA and 50 USA300 methicillin-susceptible S. aureus (MSSA) isolates were CP negative (CP−). Moreover, all 16 USA500 isolates, which have been postulated to be the progenitor lineage of USA300, were also CP−. Whole-genome sequence analysis of 146 CP− USA300 MRSA isolates revealed they all carry a cap5 locus with 4 conserved mutations compared with strain Newman. Genetic complementation experiments revealed that three of these mutations (in the cap5 promoter, cap5D nucleotide 994, and cap5E nucleotide 223) ablated CP production in USA300 and that Cap5E75 Asp, located in the coenzyme-binding domain, is essential for capsule production. All but three USA300 MSSA isolates had the same four cap5 mutations found in USA300 MRSA isolates. Most isolates with a USA500 pulsotype carried three of these four USA300-specific mutations, suggesting the fourth mutation occurred in the USA300 lineage. Phylogenetic analysis of the cap loci of our USA300 isolates as well as publicly available genomes from 41 other sequence types revealed that the USA300-specific cap5 mutations arose sequentially in S. aureus in a common ancestor of USA300 and USA500 isolates.

Streptococcus agalactiae Capsule Polymer Length and Attachment Is Determined by the Proteins CpsABCD

The production of capsular polysaccharides (CPS) or secreted exopolysaccharides is ubiquitous in bacteria, and the Wzy pathway constitutes a prototypical mechanism to produce these structures. Despite the differences in polysaccharide composition among species, a group of proteins involved in this pathway is well conserved. Streptococcus agalactiae (group B Streptococcus; GBS) produces a CPS that represents the main virulence factor of the bacterium and is a prime target in current vaccine development. We used this human pathogen to investigate the roles and potential interdependencies of the conserved proteins CpsABCD encoded in the cps operon, by developing knock-out and functional mutant strains. The mutant strains were examined for CPS quantity, size, and attachment to the cell surface as well as CpsD phosphorylation. We observed that CpsB, -C, and -D compose a phosphoregulatory system where the CpsD autokinase phosphorylates its C-terminal tyrosines in a CpsC-dependent manner. These Tyr residues are also the target of the cognate CpsB phosphatase. An interaction between CpsD and CpsC was observed, and the phosphorylation state of CpsD influenced the subsequent action of CpsC. The CpsC extracellular domain appeared necessary for the production of high molecular weight polysaccharides by influencing CpsA-mediated attachment of the CPS to the bacterial cell surface. In conclusion, although having no impact on cps transcription or the synthesis of the basal repeating unit, we suggest that these proteins are fine-tuning the last steps of CPS biosynthesis (i.e. the balance between polymerization and attachment to the cell wall).

Comparative metabolomic analysis of the effects of light quality on polysaccharide production of cyanobacterium Nostoc flagelliforme

Light quality strongly affects cell growth and polysaccharide production of cyanobacteria, however, the metabolic mechanism of polysaccharide biosynthesis upon light quality has not been fully explored yet. The effects of light quality on the metabolic changes of Nostoc flagelliforme were comprehensively investigated using red, yellow, green, blue, purple light emitting diodes for illumination. The principal component analysis revealed that red, yellow, green, blue, and purple light-grown cells were metabolically distinct from those illuminated with white fluorescent light, and the influence of light quality on metabolism was particularly dependent on light wavelength. Significant correlations between the metabolic profile and extracellular polysaccharide (EPS) and capsular polysaccharide (CPS) production were revealed by partial least-squares to latent structure analysis respectively, and analysis of key metabolites showed that CPS production was closely related with cell growth while the biosynthesis of EPS was more closely related with light conditions. Furthermore, as the influence pattern of red light on cellular metabolism and polysaccharide production was quite different from other light conditions, the effects of intensity of red light on photosynthesis and polysaccharide production were evaluated, and the results revealed that red light induced photoinhibition and therefore stimulated polysaccharide production. Manipulation of red light was thus proposed to profitably enhance polysaccharide productivity of N. flagelliforme.

Modification of the CpsA protein reveals a role in alteration of the Streptococcus agalactiae cell envelope.

The bacterial cell envelope is a crucial first line of defense for a systemic pathogen, with production of capsular polysaccharides and maintenance of the peptidoglycan cell wall serving essential roles in survival in the host environment. The LytR-CpsA-Psr proteins are important for cell envelope maintenance in many Gram-positive species. In this study, we examined the role of the extracellular domain of the CpsA protein of the zoonotic pathogen group B Streptococcus in capsule production and cell wall integrity. CpsA has multiple functional domains, including a DNA-binding/transcriptional activation domain and a large extracellular domain. We demonstrated that episomal expression of extracellularly truncated CpsA causes a dominant-negative effect on capsule production when expressed in the wild-type strain. Regions of the extracellular domain essential to this phenotype were identified. The dominant-negative effect could be recapitulated by addition of purified CpsA protein or a short CpsA peptide to cultures of wild-type bacteria. Changes in cell wall morphology were also observed when the dominant-negative peptide was added to wild-type cultures. Fluorescently labeled CpsA peptide could be visualized bound at the mid-cell region near the division septae, suggesting a novel role for CpsA in cell division. Finally, expression of truncated CpsA also led to attenuation of virulence in zebrafish models of infection, to levels below that of a cpsA deletion strain, demonstrating the key role of the extracellular domain in virulence of GBS.

Cyclic AMP and Cyclic AMP-Receptor Protein are Required for Optimal Capsular Polysaccharide Expression

Vibrio vulnificus causes fatal infections in susceptible individuals. Group 1 capsular polysaccharide (CPS) operon is responsible for CPS expression, which plays an essential role in the pathogenesis of this pathogen. Cyclic AMP (cAMP) and cAMP receptor protein (Crp) complex, which responds to glucose availability and functions as a global regulator, has been known to affect CPS production in this pathogen. This study was undertaken to experimentally verify whether cAMP-Crp directly or indirectly affects CPS production. A mutation in cyaA encoding adenylate cyclase, which is required for cAMP biosynthesis, inhibited V. vulnificus growth and changed opaque colonies to translucent colonies, and these changes were recovered by complementing cyaA or by adding exogenous cAMP. A mutation in crp encoding Crp also inhibited V. vulnificus growth and changed opaque colonies to translucent colonies, and these changes were recovered by complementing crp. Moreover, the crp or cyaA mutation decreased the susceptibility of V. vulnificus against NaOCl. The crp mutation reduced the transcription levels of group 1 CPS operon on a per cell basis. Glucose addition in the absence of Crp stimulated V. vulnificus growth, changed translucent colonies to opaque colonies, and increased the transcription levels of group 1 CPS operon. These results indicate that cAMP or Crp is indirectly involved in optimal CPS production by positively affecting metabolism or V. vulnificus growth rather than by directly controlling the expression of group 1 CPS operon.

Role of anaerobiosis in capsule production and biofilm formation in Vibrio vulnificus.

Vibrio vulnificus, a pervasive human pathogen, can cause potentially fatal septicemia after consumption of undercooked seafood. Biotype 1 strains of V. vulnificus are most commonly associated with human infection and are separated into two genotypes, clinical (C) and environmental (E), based on the virulence-correlated gene. For ingestion-based vibriosis to occur, this bacterium must be able to withstand multiple conditions as it traverses the gastrointestinal tract and ultimately gains entry into the bloodstream. One such condition, anoxia, has yet to be extensively researched in V. vulnificus. We investigated the effect of oxygen availability on capsular polysaccharide (CPS) production and biofilm formation in this bacterium, both of which are thought to be important for disease progression. We found that lack of oxygen elicits a reduction in both CPS and biofilm formation in both genotypes. This is further supported by the finding that pilA, pilD, and mshA genes, all of which encode type IV pilin proteins that aid in attachment to surfaces, were downregulated during anaerobiosis. Surprisingly, E-genotypes exhibited distinct differences in gene expression levels of capsule and attachment genes compared to C-genotypes, both aerobically and anaerobically. The importance of understanding these disparities may give insight into the observed differences in environmental occurrence and virulence potential between these two genotypes of V. vulnificus.

Staphylococcus aureus gene expression in a rat model of infective endocarditis.

BackgroundDiabetes mellitus is a frequent underlying comorbidity in patients with Staphylococcus aureus endocarditis, and it represents a risk factor for complications and a negative outcome. The pathogenesis of staphylococcal endocardial infections in diabetic hosts has been poorly characterized, and little is known about S. aureus gene expression in endocardial vegetations.MethodsWe utilized a rat model of experimental S. aureus endocarditis to compare the pathogenesis of staphylococcal infection in diabetic and nondiabetic hosts and to study the global S. aureus transcriptome in endocardial vegetations in vivo.ResultsDiabetic rats had higher levels of bacteremia and larger endocardial vegetations than nondiabetic control animals. Microarray analyses revealed that 61 S. aureus genes were upregulated in diabetic rats, and the majority of these bacterial genes were involved in amino acid and carbohydrate metabolism. When bacterial gene expression in vivo (diabetic or nondiabetic endocardial vegetations) was compared to in vitro growth conditions, higher in vivo expression of genes encoding toxins and proteases was observed. Additionally, genes involved in the production of adhesins, capsular polysaccharide, and siderophores, as well as in amino acid and carbohydrate transport and metabolism, were upregulated in endocardial vegetations. To test the contribution of selected upregulated genes to the pathogenesis of staphylococcal endocarditis, isogenic deletion mutants were utilized. A mutant defective in production of the siderophore staphyloferrin B was attenuated in the endocarditis model, whereas the virulence of a surface adhesin (ΔsdrCDE) mutant was similar to that of the parental S. aureus strain.ConclusionsOur results emphasize the relevance of diabetes mellitus as a risk factor for infectious endocarditis and provide a basis for understanding gene expression during staphylococcal infections in vivo.Electronic supplementary materialThe online version of this article (doi:10.1186/s13073-014-0093-3) contains supplementary material, which is available to authorized users.

Molecular mapping of the cell wall polysaccharides of the human pathogen Streptococcus agalactiae.

The surface of many bacterial pathogens is covered with polysaccharides that play important roles in mediating pathogen-host interactions. In Streptococcus agalactiae, the capsular polysaccharide (CPS) is recognized as a major virulence factor while the group B carbohydrate (GBC) is crucial for peptidoglycan biosynthesis and cell division. Despite the important roles of CPS and GBC, there is little information available on the molecular organization of these glycopolymers on the cell surface. Here, we use atomic force microscopy (AFM) and transmission electron microscopy (TEM) to analyze the nanoscale distribution of CPS and GBC in wild-type (WT) and mutant strains of S. agalactiae. TEM analyses reveal that in WT bacteria, peptidoglycan is covered with a very thin (few nm) layer of GBC (the "pellicle") overlaid by a 15-45 nm thick layer of CPS (the "capsule"). AFM-based single-molecule mapping with specific antibody probes shows that CPS is exposed on WT cells, while it is hardly detected on mutant cells impaired in CPS production (ΔcpsE mutant). By contrast, both TEM and AFM show that CPS is over-expressed in mutant cells altered in GBC expression (ΔgbcO mutant), indicating that the production of the two surface glycopolymers is coordinated in WT cells. In addition, AFM topographic imaging and molecular mapping with specific lectin probes demonstrate that removal of CPS (ΔcpsE), but not of GBC (ΔgbcO), leads to the exposure of peptidoglycan, organized into 25 nm wide bands running parallel to the septum. These results indicate that CPS forms a homogeneous barrier protecting the underlying peptidoglycan from environmental exposure, while the presence of GBC does not prevent peptidoglycan detection. This work shows that single-molecule AFM, combined with high-resolution TEM, represents a powerful platform for analysing the molecular arrangement of the cell wall polymers of bacterial pathogens.

Acyl-homoserine lactone recognition and response hindering the quorum-sensing regulator EsaR.

During quorum sensing in the plant pathogen Pantoea stewartii subsp. stewartii, EsaI, an acyl-homoserine lactone (AHL) synthase, and the transcription factor EsaR coordinately control capsular polysaccharide production. The capsule is expressed only at high cell density when AHL levels are high, leading to inactivation of EsaR. In lieu of detailed structural information, the precise mechanism whereby EsaR recognizes AHL and is hindered by it, in a response opposite to that of most other LuxR homologues, remains unresolved. Hence, a random mutagenesis genetic approach was designed to isolate EsaR* variants that are immune to the effects of AHL. Error-prone PCR was used to generate the desired mutants, which were subsequently screened for their ability to repress transcription in the presence of AHL. Following sequencing, site-directed mutagenesis was used to generate all possible mutations of interest as single, rather than multiple amino acid substitutions. Eight individual amino acids playing a critical role in the AHL-insensitive phenotype have been identified. The ability of EsaR* variants to bind AHL and the effect of individual substitutions on the overall conformation of the protein were examined through in vitro assays. Six EsaR* variants had a decreased ability to bind AHL. Fluorescence anisotropy was used to examine the relative DNA binding affinity of the final two EsaR* variants, which retained some AHL binding capability but remained unresponsive to it, perhaps due to an inability of the N-terminal domain to transduce information to the C-terminal domain.

Increased proliferation and decreased membrane permeability as defense mechanisms of Fusobacterium nucleatum against human neutrophilic peptide-1

Human neutrophilic peptides (HNPs) constitute a class of host defense molecules, which contribute to the non-oxidative killing of bacteria and other microorganisms. Since the adaptability is crucial to bacterial survival in changing environments, it is of interest to know how Fusobacterium nucleatum, the major bridge organism connecting early and late colonizers in dental biofilms, defends itself against HNPs. This study aimed to examine the planktonic growth, membrane permeability, and biofilm formation characteristics as defense mechanisms of F. nucleatum against HNP-1. In all experiments, the type strain of F. nucleatum (ssp. nucleatum ATCC 25586) and two clinical strains (ssp. nucleatum AHN 9508 and ssp. polymorphum AHN 9910) were used. Planktonic growth (measured in colony forming units), capsular polysaccharide production (visualized by Ziehl-Neelsen stain), membrane permeability (demonstrated as N-phenyl-1-naphthylamine uptake), biofilm formation, and established biofilm development (measured as total mass and polysaccharide levels) were analyzed in the presence of 0 μg/ml (control), 1 μg/ml, 5 μg/ml, and 10 μg/ml of HNP-1. Planktonic growth of the strains AHN 9508 and ATCC 25586 were significantly (p < 0.05) increased in the presence of HNP-1, while their membrane permeability decreased (p < 0.005) in the planktonic form. HNP-1 decreased the biofilm formation of the strains ATCC 25586 and AHN 9910, whereas it increased the growth of the strain AHN 9508 in established biofilms. Capsule formation and polysaccharide production were not observed in any strain. We conclude that the inhibition of the membrane permeability and the increase in planktonic and established biofilm growth could act as bacterial defense mechanisms against neutrophilic defensins. In addition, this strain-dependent survival ability against HNP-1 may explain the variation in the virulence of different F. nucleatum strains.

Isolation and characterization of a novel Acidithiobacillus ferrivorans strain from the Chilean Altiplano: attachment and biofilm formation on pyrite at low temperature

Microorganisms are used to aid the extraction of valuable metals from low-grade sulfide ores in mines worldwide, but relatively little is known about this process in cold environments. This study comprises a preliminary analysis of the bacterial diversity of the polyextremophilic acid River Aroma located in the Chilean Altiplano, and revealed that Betaproteobacteria was the most dominant bacterial group (Gallionella-like and Thiobacillus-like). Taxa characteristic of leaching environments, such Acidithiobacillus and Leptospirillum, were detected at low abundances. Also, bacteria not associated with extremely acidic, metal-rich environments were found. After enrichment in iron- and sulfur-oxidizing media, we isolated and identified a novel psychrotolerant Acidithiobacillus ferrivorans strain ACH. This strain can grow using ferrous iron, sulfur, thiosulfate, tetrathionate and pyrite, as energy sources. Optimal growth was observed in the presence of pyrite, where cultures reached a cell number of 6.5·107 cells mL−1. Planktonic cells grown with pyrite showed the presence of extracellular polymeric substances (10 °C and 28 °C), and a high density of cells attached to pyrite grains were observed at 10 °C by electron microscopy. The attachment of cells to pyrite coupons and the presence of capsular polysaccharides were visualized by using epifluorescence microscopy, through nucleic acid and lectin staining with Syto®9 and TRITC-Con A, respectively. Interestingly, we observed high cell adhesion including the formation of microcolonies within 21 days of incubation at 4 °C, which was correlated with a clear induction of capsular polysaccharides production. Our data suggests that attachment to pyrite is not temperature-dependent in At. ferrivorans ACH. The results of this study highlight the potential of this novel psychrotolerant strain in oxidation and attachment to minerals under low-temperature conditions.

Improved capsular polysaccharide production by Streptococcus pneumoniae serotype 14 using continuous cultivation

The capsular polysaccharide (PS) is the most important pneumococcal virulence factor and is currently used as antigen in all pneumococcal vaccines. Despite its physiological and epidemiological importance, meager studies have been devoted to improve PS production and understand its relationship with pneumococcal central metabolism. In this study, kinetics of growth and production of PS by Streptococcus pneumoniae serotype 14 (PS14) in batch and continuous cultivation were investigated. Strong cell lysis was observed in batch cultivation, while accumulation of organic acids and autolysis was avoided in continuous cultivation. In the continuous cultivation was possible to achieve higher concentration of biomass and PS14. Calculation of kinetic parameters demonstrated that PS14 is a cell-associated product. The coefficients for growth-associated stoichiometric true yield and maintenance were determined as 0.25gglucosegbiomass−1 and 1.24gglucose(gbiomassh)−1, respectively. The maximum productivity of PS14 released in the supernatant (PS14R) and cell-bound PS14 (PS14C) were obtained at a dilution rate of 0.8h−1, respectively, 85 and 122mg (gbiomassh)−1. Compared to batch fermentation, both PS14R and PS14C productivities were increased by about 300% in the continuous process. These findings demonstrate that continuous cultivation is a promising strategy for PS production to be used in pneumococcal vaccines.

Genetic Analysis and Prevalence Studies of the brp Exopolysaccharide Locus of Vibrio vulnificus

Phase variation in the Gram-negative human pathogen Vibrio vulnificus involves three colonial morphotypes- smooth opaque colonies due to production of capsular polysaccharide (CPS), smooth translucent colonies as the result of little or no CPS expression, and rugose colonies due to production of a separate extracellular polysaccharide (EPS), which greatly enhances biofilm formation. Previously, it was shown that the brp locus, which consists of nine genes arranged as an operon, is up-regulated in rugose strains in a c-di-GMP-dependent manner, and that plasmid insertions into the locus resulted in loss of rugosity and efficient biofilm production. Here, we have used non-polar mutagenesis to assess the involvement of individual brp genes in production of EPS and related phenotypes. Inactivation of genes predicted to be involved in various stages of EPS biosynthesis eliminated both the rugose colonial appearance and production of EPS, while knockout of a predicted flippase function involved in EPS transport resulted in a dry, lightly striated phenotype, which was associated with a reduction of brp-encoded EPS on the cell surface. All brp mutants retained the reduced motility characteristic of rugose strains. Lastly, we provide evidence that the brp locus is highly prevalent among strains of V. vulnificus.