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1.  Characterization of the spore-forming Bacillus cereus sensu lato group and Clostridium perfringens bacteria isolated from the Australian dairy farm environment 
BMC Microbiology  2015;15:38.
The Bacillus cereus sensu lato group and Clostridium perfringens are spore-forming bacteria often associated with food spoilage and which can cause emetic and diarrheal syndromes in humans and ruminants. This study characterised the phenotypes and genotypes of 50 Bacillus cereus s. l. isolates and 26 Clostridium perfringens isolates from dairy farms environments in Victoria, Australia.
Five of the seven B. cereus s. l. species were isolated, and analysis of the population diversity using Pulsed-Field Gel Electrophoresis (PFGE) suggested that the populations are largely distinct to each farm. Enterotoxin production by representative isolates of each B. cereus s. l. species identified was typically found to be reduced in milk, compared with broth. Among the C. perfringens isolates, only two different toxin types were identified, type A and D. Bovine and ovine farms harbored only type A whereas both type A and D were found on two of the three caprine farms.
This study showed that the B. cereus s. l. populations on the sampled farms exhibit a broad diversity in both species and genotypes. The risk of toxin-induced diarrheal illness through consumption of contaminated milk may be limited, in comparison with other food matrices. Type A strains of C. perfringens were the most abundant on dairy farms in Victoria, however type D may be of concern on caprine farms as it can cause enterotoxemia in goats.
Electronic supplementary material
The online version of this article (doi:10.1186/s12866-015-0377-9) contains supplementary material, which is available to authorized users.
PMCID: PMC4336692
Bacillus cereus sensu lato; Clostridium perfringens; Dairy farm; PFGE; Toxin
2.  MoMyb1 is required for asexual development and tissue-specific infection in the rice blast fungus Magnaporthe oryzae 
BMC Microbiology  2015;15:37.
The Myb super-family of proteins contain a group of functionally diverse transcriptional activators found in plant, animal and fungus. Myb proteins are involved in cell proliferation, differentiation and apoptosis, and have crucial roles in telomeres. The purpose of this study was to characterize the biological function of Myb1 protein in the rice blast fungus Magnaporthe oryzae.
We identified the Saccharomyces cerevisiae BAS1 homolog MYB1 in M. oryzae, named MoMyb1. MoMyb1 encodes a protein of 322 amino acids and has two SANT domains and is well conserved in various organisms. Targeted gene deletion of MoMYB1 resulted in a significant reduction in vegetative growth and showed defects in conidiation and conidiophore development. Quantitative RT-PCR analysis revealed that the transcription levels of several conidiophore-related genes were apparently decreased in the ΔMomyb1 mutant. Inoculation with mycelia mats displayed that the virulence of the ΔMomyb1 mutant was not changed on rice leaves but was non-pathogenic on rice roots in comparison to the wild type Guy11. In addition, ∆Momyb1 mutants showed increased resistance to osmotic stresses but more sensitive to cell wall stressor calcofluor white (CFW). Further analysis revealed that MoMyb1 has an important role in the cell wall biosynthesis pathway.
This study provides the evidence that MoMyb1 is a key regulator involved in conidiogenesis, stress response, cell wall integrity and pathogenesis on rice roots in the filamentous phytopathogen M. oryzae.
Electronic supplementary material
The online version of this article (doi:10.1186/s12866-015-0375-y) contains supplementary material, which is available to authorized users.
PMCID: PMC4336695
Magnaporthe oryzae; Conidiogenesis; Stress response; Cell wall integrity; Pathogenesis
3.  An extracytoplasmic function sigma factor-dependent periplasmic glutathione peroxidase is involved in oxidative stress response of Shewanella oneidensis 
BMC Microbiology  2015;15:34.
Bacteria use alternative sigma factors (σs) to regulate condition-specific gene expression for survival and Shewanella harbors multiple ECF (extracytoplasmic function) σ genes and cognate anti-sigma factor genes. Here we comparatively analyzed two of the rpoE-like operons in the strain MR-1: rpoE-rseA-rseB-rseC and rpoE2-chrR.
RpoE was important for bacterial growth at low and high temperatures, in the minimal medium, and high salinity. The degP/htrA orthologue, required for growth of Escherichia coli and Pseudomonas aeruginosa at high temperature, is absent in Shewanella, while the degQ gene is RpoE-regulated and is required for bacterial growth at high temperature. RpoE2 was essential for the optimal growth in oxidative stress conditions because the rpoE2 mutant was sensitive to hydrogen peroxide and paraquat. The operon encoding a ferrochelatase paralogue (HemH2) and a periplasmic glutathione peroxidase (PgpD) was identified as RpoE2-dependent. PgpD exhibited higher activities and played a more important role in the oxidative stress responses than the cytoplasmic glutathione peroxidase CgpD under tested conditions. The rpoE2-chrR operon and the identified regulon genes, including pgpD and hemH2, are coincidently absent in several psychrophilic and/or deep-sea Shewanella strains.
In S. oneidensis MR-1, the RpoE-dependent degQ gene is required for optimal growth under high temperature. The rpoE2 and RpoE2-dependent pgpD gene encoding a periplasmic glutathione peroxidase are involved in oxidative stress responses. But rpoE2 is not required for bacterial growth at low temperature and it even affected bacterial growth under salt stress, indicating that there is a tradeoff between the salt resistance and RpoE2-mediated oxidative stress responses.
Electronic supplementary material
The online version of this article (doi:10.1186/s12866-015-0357-0) contains supplementary material, which is available to authorized users.
PMCID: PMC4336711
Periplasmic glutathione peroxidase; Shewanella; ECF sigma factor; Oxidative stress response
4.  Antibiosis interaction of Staphylococccus aureus on Aspergillus fumigatus assessed in vitro by mixed biofilm formation 
BMC Microbiology  2015;15:33.
Microorganisms of different species interact in several ecological niches, even causing infection. During the infectious process, a biofilm of single or multispecies can develop. Aspergillus fumigatus and Staphyloccocus aureus are etiologic agents that can cause infectious keratitis. We analyzed in vitro single A. fumigatus and S. aureus, and mixed A. fumigatus-S. aureus biofilms. Both isolates were from patients with infectious keratitis. Structure of the biofilms was analyzed through microscopic techniques including scanning electron microscopy (SEM), transmission electron microscopy (TEM), confocal, and fluorescence microscopy (CLSM) in mixed biofilm as compared with the single A. fumigatus biofilm.
To our knowledge, this is the first time that the structural characteristics of the mixed biofilm A. fumigatus-A. fumigatus were described and shown. S. aureus sharply inhibited the development of biofilm formed by A. fumigatus, regardless of the stage of biofilm formation and bacterial inoculum. Antibiosis effect of bacterium on fungus was as follows: scarce production of A. fumigatus biofilm; disorganized fungal structures; abortive hyphae; and limited hyphal growth; while conidia also were scarce, have modifications in their surface and presented lyses. Antagonist effect did not depend on bacterial concentration, which could probably be due to cell-cell contact interactions and release of bacterial products. In addition, we present images about the co-localization of polysaccharides (glucans, mannans, and chitin), and DNA that form the extracellular matrix (ECM). In contrast, single biofilms showed extremely organized structures: A. fumigatus showed abundant hyphal growth, hyphal anastomosis, and channels, as well as some conidia, and ECM. S. aureus showed microcolonies and cell-to-cell bridges and ECM.
Herein we described the antibiosis relationship of S. aureus against A. fumigatus during in vitro biofilm formation, and report the composition of the ECM formed.
PMCID: PMC4335557
Biofilm; Extracellular matrix; Antibiosis of Staphylococcus aureus against Aspergillus fumigatus; Fungus-bacteria interaction
5.  Lipopeptide biosynthesis in Pseudomonas fluorescens is regulated by the protease complex ClpAP 
BMC Microbiology  2015;15:29.
Lipopeptides (LP) are structurally diverse compounds with potent surfactant and broad-spectrum antibiotic activities. In Pseudomonas and other bacterial genera, LP biosynthesis is governed by large multimodular nonribosomal peptide synthetases (NRPS). To date, relatively little is known about the regulatory genetic network of LP biosynthesis.
This study provides evidence that the chaperone ClpA, together with the serine protease ClpP, regulates the biosynthesis of the LP massetolide in Pseudomonas fluorescens SS101. Whole-genome transcriptome analyses of clpA and clpP mutants showed their involvement in the transcription of the NRPS genes massABC and the transcriptional regulator massAR. In addition, transcription of genes associated with cell wall and membrane biogenesis, energy production and conversion, amino acid transport and metabolism, and pilus assembly were altered by mutations in clpA and clpP. Proteome analysis allowed the identification of additional cellular changes associated to clpA and clpP mutations. The expression of proteins of the citrate cycle and the heat shock proteins DnaK and DnaJ were particularly affected. Combined with previous findings, these results suggest that the ClpAP complex regulates massetolide biosynthesis via the pathway-specific, LuxR-type regulator MassAR, the heat shock proteins DnaK and DnaJ, and proteins of the TCA cycle.
Combining transcriptome and proteome analyses provided new insights into the regulation of LP biosynthesis in P. fluorescens and led to the identification of specific missing links in the regulatory pathways.
Electronic supplementary material
The online version of this article (doi:10.1186/s12866-015-0367-y) contains supplementary material, which is available to authorized users.
PMCID: PMC4332742
6.  Biochemical and genetic diversity of carbohydrate-fermenting and obligate amino acid-fermenting hyper-ammonia-producing bacteria from Nellore steers fed tropical forages and supplemented with casein 
BMC Microbiology  2015;15:28.
Dietary protein plays a major role in ruminant nutrition, and protein supplementation is a widespread practice among farmers in the tropics. Ruminal bacteria are the main agents of dietary protein and amino acid degradation, yet few studies have focused on the isolation and characterization of hyper-ammonia-producing bacteria in animals fed tropical diets or supplemented with rumen-degradable proteins. This work investigated the bacterial community diversity of the rumen of Nellore steers fed tropical forages, with or without casein supplementation. We also isolated and characterized ruminal bacteria showing high levels of ammonia production.
Polymerase chain reaction–denaturing gradient gel electrophoresis analysis indicated no differences in the ruminal bacterial community composition between the control and supplemented animals. Amino acid-fermenting bacteria (n = 250) were isolated from crossbred Nellore steers fed Tifton 85 (Cynodon sp.) using trypticase as the sole carbon and organic nitrogen source in the enrichment and isolation media. The deamination rates in isolates obtained from steers supplemented with casein showed a higher incidence of deamination rates >350 nmol NH3 mg protein−1 min−1 (P < 0.05), whereas isolates obtained from steers without supplementation showed deamination rates <200 nmol NH3 mg protein−1 min−1. Although most isolates (84%) could ferment carbohydrates, none could hydrolyze proteins or use urea to sustain growth. All isolates were sensitive to lasalocid and monensin (1 μmol l−1), and similarity analysis of the 16S rRNA sequences indicated a predominance of bacteria from the order Clostridiales, with variable homology (73–99%) to known bacterial species.
These results expand what is known about the biochemical and genetic diversity of hyper-ammonia-producing bacteria, and emphasize the role of carbohydrate-fermenting bacteria in ammonia production in the rumen.
Electronic supplementary material
The online version of this article (doi:10.1186/s12866-015-0369-9) contains supplementary material, which is available to authorized users.
PMCID: PMC4332921
Deamination; Clostridiales; ionophores; dietary protein; PCR-DGGE
7.  Structure and function of a short LOV protein from the marine phototrophic bacterium Dinoroseobacter shibae 
BMC Microbiology  2015;15:30.
Light, oxygen, voltage (LOV) domains are widely distributed in plants, algae, fungi, bacteria, and represent the photo-responsive domains of various blue-light photoreceptor proteins. Their photocycle involves the blue-light triggered adduct formation between the C(4a) atom of a non-covalently bound flavin chromophore and the sulfur atom of a conserved cysteine in the LOV sensor domain. LOV proteins show considerable variation in the structure of N- and C-terminal elements which flank the LOV core domain, as well as in the lifetime of the adduct state.
Here, we report the photochemical, structural and functional characterization of DsLOV, a LOV protein from the photoheterotrophic marine α-proteobacterium Dinoroseobacter shibae which exhibits an average adduct state lifetime of 9.6 s at 20°C, and thus represents the fastest reverting bacterial LOV protein reported so far. Mutational analysis in D. shibae revealed a unique role of DsLOV in controlling the induction of photopigment synthesis in the absence of blue-light. The dark state crystal structure of DsLOV determined at 1.5 Å resolution reveals a conserved core domain with an extended N-terminal cap. The dimer interface in the crystal structure forms a unique network of hydrogen bonds involving residues of the N-terminus and the β-scaffold of the core domain. The structure of photoexcited DsLOV suggests increased flexibility in the N-cap region and a significant shift in the Cα backbone of β strands in the N- and C-terminal ends of the LOV core domain.
The results presented here cover the characterization of the unusual short LOV protein DsLOV from Dinoroseobacter shibae including its regulatory function, extremely fast dark recovery and an N-terminus mediated dimer interface. Due to its unique photophysical, structural and regulatory properties, DsLOV might thus serve as an alternative model system for studying light perception by LOV proteins and physiological responses in bacteria.
Electronic supplementary material
The online version of this article (doi:10.1186/s12866-015-0365-0) contains supplementary material, which is available to authorized users.
PMCID: PMC4335406
Aerobic anoxygenic photosynthesis; Blue-light photoreceptor; Crystal structure; Dimerization; Dinoroseobacter shibae; LOV domain; PAS domain; Photocycle
8.  CsrA impacts survival of Yersinia enterocolitica by affecting a myriad of physiological activities 
BMC Microbiology  2015;15:31.
A previous study identified a Yersinia enterocolitica transposon mutant, GY448, that was unable to export the flagellar type three secretion system (T3SS)-dependent phospholipase, YplA. This strain was also deficient for motility and unable to form colonies on Lauria-Bertani agar medium. Preliminary analysis suggested it carried a mutation in csrA. CsrA in Escherichia coli is an RNA-binding protein that is involved in specific post-transcriptional regulation of a myriad of physiological activities. This study investigated how CsrA affects expression of the flagellar regulatory cascade that controls YplA export and motility. It also explored the effect of csrA mutation on Y. enterocolitica in response to conditions that cue physiological changes important for growth in environments found both in nature and the laboratory.
The precise location of the transposon insertion in GMY448 was mapped within csrA. Genetic complementation restored disruptions in motility and the YplA export phenotype (Yex), which confirmed this mutation disrupted CsrA function. Mutation of csrA affected expression of yplA and flagellar genes involved in flagellar T3SS dependent export and motility by altering expression of the master regulators flhDC. Mutation of csrA also resulted in increased sensitivity of Y. enterocolitica to various osmolytes, temperatures and antibiotics.
The results of this study reveal unique aspects of how CsrA functions in Y. enterocolitica to control its physiology. This provides perspective on how the Csr system is susceptible to adaptation to particular environments and bacterial lifestyles.
PMCID: PMC4336687
Yersinia; CsrA; Csr system; Motility; Salt sensitivity; Antibiotic sensitivity; Temperature sensitivity; Psychrotroph; Mutant selection
9.  New insights into the fungal community from the raw genomic sequence data of fig wasp Ceratosolen solmsi 
BMC Microbiology  2015;15(1):27.
To date, biologists have discovered a large amount of valuable information from assembled genomes, but the abundant microbial data that is hidden in the raw genomic sequence data of plants and animals is usually ignored. In this study, the richness and composition of fungal community were determined in the raw genomic sequence data of Ceratosolen solmsi (RGSD-CS).
To avoid the interference from sequences of C. solmsi, the unmapped raw data (about 17.1%) was obtained by excluding the assembled genome of C. solmsi from RGSD-CS. Comparing two fungal reference datasets, internal transcribed spacer (ITS) and large ribosomal subunit (LSU) of rRNA, the ITS dataset discovered a more diverse fungal community and was therefore selected as the reference dataset for evaluating the fungal community based on the unmapped raw data. The threshold of 95% sequence identity revealed many more matched fungal reads and fungal richness in the unmapped raw data than those by identities above 95%. Based on the threshold of 95% sequence identity, the fungal community of RGSD-CS was primarily composed of Saccharomycetes (88.4%) and two other classes (Agaricomycetes and Sordariomycetes, 8.3% in total). Compared with the fungal community of other reported fig wasps, Agaricomycetes and Eurotiomycetes were found to be unique to C. solmsi. In addition, the ratio of total fungal reads to RGSD-CS was estimated to be at least 4.8 × 10−3, which indicated that a large amount of fungal data was contained in RGSD-CS. However, rarefaction measure indicated that a deeper sequencing coverage with RGSD-CS was required to discover the entire fungal community of C. solmsi.
This study investigated the richness and composition of fungal community in RGSD-CS and provided new insights into the efficient study of microbial diversity using raw genomic sequence data.
Electronic supplementary material
The online version of this article (doi:10.1186/s12866-015-0370-3) contains supplementary material, which is available to authorized users.
PMCID: PMC4329198
Fungal community; Unmapped raw data; Fig wasp; Fungal reference datasets
10.  CrdR function in a curdlan-producing Agrobacterium sp. ATCC31749 strain 
BMC Microbiology  2015;15(1):25.
Agrobacterium sp. ATCC31749 is an efficient curdlan producer at low pH and under nitrogen starvation. The helix-turn-helix transcriptional regulatory protein (crdR) essential for curdlan production has been analyzed, but whether crdR directly acts to cause expression of the curdlan biosynthesis operon (crdASC) is uncertain. To elucidate the molecular function of crdR in curdlan biosynthesis, we constructed a crdR knockout mutant along with pBQcrdR and pBQNcrdR vectors with crdR expression driven by a T5 promoter and crdR native promoter, respectively. Also, we constructed a pAG with the green fluorescent protein (GFP) gene driven by a curdlan biosynthetic operon promoter (crdP) to measure the effects of crdR expression on curdlan biosynthesis.
Compared with wild-type (WT) strain biomass production, the biomass of the crdR knockout mutant was not significantly different in either exponential or stationary phases of growth. Mutant cells were non-capsulated and planktonic and produced significantly less curdlan. WT cells were curdlan-capsulated and aggregated in the stationery phase. pBQcrdR transformed to the WT strain had a 38% greater curdlan yield and pBQcrdR and pBQNcrdR transformed to the crdR mutant strain recovered 18% and 105% curdlan titers of the WT ATCC31749 strain, respectively. Consistent with its function of promoting curdlan biosynthesis, curdlan biosynthetic operon promoter (crdP) controlled GFP expression caused the transgenic strain to have higher GFP relative fluorescence in the WT strain, and no color change was observed with low GFP relative fluorescence in the crdR mutant strain as evidenced by fluorescent microscopy and spectrometric assay. q-RT-PCR revealed that crdR expression in the stationary phase was greater than in the exponential phase, and crdR overexpression in the WT strain increased crdA, crdS, and crdC expression. We also confirmed that purified crdR protein can specifically bind to the crd operon promoter region, and we inferred that crdR directly acts to cause expression of the curdlan biosynthesis operon (crdASC).
CrdR is a positive transcriptional regulator of the crd operon for promoting curdlan biosynthesis in ATCC31749. The potential binding region of crdR is located within the −98 bp fragment upstream from the crdA start codon
Electronic supplementary material
The online version of this article (doi:10.1186/s12866-015-0356-1) contains supplementary material, which is available to authorized users.
PMCID: PMC4327974
crdR; Curdlan; Agrobacterium; Transcriptional regulator
11.  Phenotypic and genotypic characterization of Staphylococci causing breast peri-implant infections in oncologic patients 
BMC Microbiology  2015;15(1):26.
Staphylococcus epidermidis and S. aureus have been identified as the most common bacteria responsible for sub-clinical and overt breast implant infections and their ability to form biofilm on the implant as been reported as the essential factor in the development of this type of infections. Biofilm formation is a complex process with the participation of several distinct molecules, whose relative importance in different clinical settings has not yet been fully elucidated. To our knowledge this is the first study aimed at characterizing isolates causing breast peri-implant infections.
Thirteen S. aureus and seven S. epidermidis causing breast peri-implant infections were studied.
Using the broth microdilution method and the E-test, the majority of the strains were susceptible to all antibiotics tested. Methicillin resistance was detected in two S. epidermidis. All strains had different RAPD profiles and were able to produce biofilms in microtitre plate assays but, while all S. aureus carried and were able to express icaA and icaD genes, this was only true for one S. epidermidis. Biofilm development was glucose- and NaCl-induced (5 S. aureus and 1 S. epidermidis) or glucose-induced (the remaining strains). Proteinase K and sodium metaperiodate treatment had different effects on biofilms dispersion revealing that the strains studied were able to produce chemically different types of extracellular matrix mediating biofilm formation.
All S. aureus strains harboured and expressed the atlA, clfA, FnA, eno and cna genes and the majority also carried and expressed the sasG (10/13), ebpS (10/13) genes.
All S. epidermidis strains harboured and expressed the atlE, aae, embp genes, and the majority (six strains) also carried and expressed the fbe, aap genes.
Genes for S. aureus capsular types 5 and 8 were almost equally distributed. The only leukotoxin genes detected were lukE/lukD (6/13).
S. aureus and S. epidermidis breast peri-implant infections are caused by heterogeneous strains with different biofilm development mechanisms.
Since the collagen adhesin (cna) gene is not ubiquitously distributed among S. aureus, this protein could have an important role in the cause of breast peri-implant infections.
Electronic supplementary material
The online version of this article (doi:10.1186/s12866-015-0368-x) contains supplementary material, which is available to authorized users.
PMCID: PMC4328704
Implant infections; Biofilm; Staphylococcal infections; ica gene; biofilm-related genes; MSCRAMMs
12.  Development and pyrosequencing analysis of an in-vitro oral biofilm model 
BMC Microbiology  2015;15:24.
Dental caries and periodontal disease are the commonest bacterial diseases of man and can result in tooth loss. The principal method of prevention is the mechanical removal of dental plaque augmented by active agents incorporated into toothpastes and mouthrinses. In-vitro assays that include complex oral bacterial biofilms are required to accurately predict the efficacy of novel active agents in vivo. The aim of this study was to develop an oral biofilm model using the Calgary biofilm device (CBD) seeded with a natural saliva inoculum and analysed by next generation sequencing. The specific objectives were to determine the reproducibility and stability of the model by comparing the composition of the biofilms over time derived from (i) the same volunteers at different time points, and (ii) different panels of volunteers.
Pyrosequencing yielded 280,093 sequences with a mean length of 432 bases after filtering. A mean of 320 and 250 OTUs were detected in pooled saliva and biofilm samples, respectively. Principal coordinates analysis (PCoA) plots based on community membership and structure showed that replicate biofilm samples were highly similar and clustered together. In addition, there were no significant differences between biofilms derived from the same panel at different times using analysis of molecular variance (AMOVA). There were significant differences between biofilms from different panels (AMOVA, P < 0.002). PCoA revealed that there was a shift in biofilm composition between seven and 14 days (AMOVA, P < 0.001). Veillonella parvula, Veillonella atypica/dispar/parvula and Peptostreptococcus stomatis were the predominant OTUs detected in seven-day biofilms, whilst Prevotella oralis, V. parvula and Streptococcus constellatus were predominant in 14-day biofilms.
Diverse oral biofilms were successfully grown and maintained using the CBD. Biofilms derived from the same panel of volunteers were highly reproducible. This model could be used to screen both antimicrobial-containing oral care products and also novel approaches aiming to modify plaque composition, such as pre- or probiotics.
Electronic supplementary material
The online version of this article (doi:10.1186/s12866-015-0364-1) contains supplementary material, which is available to authorized users.
PMCID: PMC4332733
16S rRNA; Bacteria; Saliva; Plaque; Microbiome; Microbiota
13.  Cleavage of the moaX-encoded fused molybdopterin synthase from Mycobacterium tuberculosis is necessary for activity 
BMC Microbiology  2015;15(1):22.
Molybdopterin cofactor (MoCo) biosynthesis in Mycobacterium tuberculosis is associated with a multiplicity of genes encoding several enzymes in the pathway, including the molybdopterin (MPT) synthase, a hetero tetramer comprising two MoaD and two MoaE subunits. In addition to moaD1, moaD2, moaE1, moaE2, the M. tuberculosis genome also contains a moaX gene which encodes an MPT-synthase in which the MoaD and MoaE domains are located on a single polypeptide. In this study, we assessed the requirement for post-translational cleavage of MoaX for functionality of this novel, fused MPT synthase and attempted to establish a functional hierarchy for the various MPT-synthase encoding genes in M. tuberculosis.
Using a heterologous Mycobacterium smegmatis host and the activity of the MoCo-dependent nitrate reductase, we confirmed that moaD2 and moaE2 from M. tuberculosis together encode a functional MPT synthase. In contrast, moaD1 displayed no functionality in this system, even in the presence of the MoeBR sulphurtransferase, which contains the rhodansese-like domain, predicted to activate MoaD subunits. We demonstrated that cleavage of MoaX into its constituent MoaD and MoaE subunits was required for MPT synthase activity and confirmed that cleavage occurs between the Gly82 and Ser83 residues in MoaX. Further analysis of the Gly81-Gly82 motif confirmed that both of these residues are necessary for catalysis and that the Gly81 was required for recognition/cleavage of MoaX by an as yet unidentified protease. In addition, the MoaE component of MoaX was able to function in conjunction with M. smegmatis MoaD2 suggesting that cleavage of MoaX renders functionally interchangeable subunits. Expression of MoaX in E. coli revealed that incorrect post-translational processing is responsible for the lack of activity of MoaX in this heterologous host.
There is a degree of functional interchangeability between the MPT synthase subunits of M. tuberculosis. In the case of MoaX, post-translational cleavage at the Gly82 residue is required for function.
Electronic supplementary material
The online version of this article (doi:10.1186/s12866-015-0355-2) contains supplementary material, which is available to authorized users.
PMCID: PMC4326299  PMID: 25651977
MoCo biosynthesis; Molybdopterin synthase; mycobacteria; MoaX; MoaD; MoaE; Nitrate reductase
14.  Transcriptome profiling of Bacillus subtilis OKB105 in response to rice seedlings 
BMC Microbiology  2015;15(1):21.
Plant growth-promoting rhizobacteria (PGPR) are soil beneficial microorganisms that colonize plant roots for nutritional purposes and accordingly benefit plants by increasing plant growth or reducing disease. However, the mechanisms and pathways involved in the interactions between PGPR and plants remain unclear. In order to better understand these complex plant-PGPR interactions, changes in the transcriptome of the typical PGPR Bacillus subtilis in response to rice seedlings were analyzed.
Microarray technology was used to study the global transcriptionl response of B. subtilis OKB105 to rice seedlings after an interaction period of 2 h. A total of 176 genes representing 3.8% of the B. subtilis strain OKB105 transcriptome showed significantly altered expression levels in response to rice seedlings. Among these, 52 were upregulated, the majority of which are involved in metabolism and transport of nutrients, and stress responses, including araA, ywkA, yfls, mtlA, ydgG et al. The 124 genes that were downregulated included cheV, fliL, spmA and tua, and these are involved in chemotaxis, motility, sporulation and teichuronic acid biosynthesis, respectively.
We present a transcriptome analysis of the bacteria Bacillus subtilis OKB105 in response to rice seedings. Many of the 176 differentially expressed genes are likely to be involved in the interaction between Gram-positive bacteria and plants.
PMCID: PMC4326333  PMID: 25651892
Bacillus subtilis; Oryza sativa; Plant-microbe interactions; Transcriptomics; Microarray; Functional annotation
15.  Uniparental mitochondrial DNA inheritance is not affected in Ustilago maydis Δatg11 mutants blocked in mitophagy 
BMC Microbiology  2015;15(1):23.
Maternal or uniparental inheritance (UPI) of mitochondria is generally observed in sexual eukaryotes, however, the underlying mechanisms are diverse and largely unknown. Recently, based on the use of mutants blocked in autophagy, it has been demonstrated that autophagy is required for strict maternal inheritance in the nematode Caenorhabditis elegans. Uniparental mitochondrial DNA (mtDNA) inheritance has been well documented for numerous fungal species, and in particular, has been shown to be genetically governed by the mating-type loci in the isogamous species Cryptococcus neoformans, Phycomyces blakesleeanus and Ustilago maydis. Previously, we have shown that the a2 mating-type locus gene lga2 is decisive for UPI during sexual development of U. maydis. In axenic culture, conditional overexpression of lga2 triggers efficient loss of mtDNA as well as mitophagy. To assess a functional relationship, we have investigated UPI in U. maydis Δatg11 mutants, which are blocked in mitophagy.
This study has revealed that Δatg11 mutants are not affected in pathogenic development and this has allowed us to analyse UPI under comparable developmental conditions between mating-compatible wild-type and mutant strain combinations. Explicitly, we have examined two independent strain combinations that gave rise to different efficiencies of UPI. We demonstrate that in both cases UPI is atg11-independent, providing evidence that mitophagy is not critical for UPI in U. maydis, even under conditions of strict UPI.
Until now, analysis of a role of mitophagy in UPI has not been reported for microbial species. Our study suggests that selective autophagy does not contribute to UPI in U. maydis, but is rather a consequence of selective mtDNA elimination in response to mitochondrial damage.
Electronic supplementary material
The online version of this article (doi:10.1186/s12866-015-0358-z) contains supplementary material, which is available to authorized users.
PMCID: PMC4326477  PMID: 25652096
Mitophagy; Uniparental mitochondrial inheritance; mtDNA; Ustilago maydis; Fungi; Atg11
16.  Transient transformation of Podosphaera xanthii by electroporation of conidia 
BMC Microbiology  2015;15(1):20.
Powdery mildew diseases are a major phytosanitary issue causing important yield and economic losses in agronomic, horticultural and ornamental crops. Powdery mildew fungi are obligate biotrophic parasites unable to grow on culture media, a fact that has significantly limited their genetic manipulation. In this work, we report a protocol based on the electroporation of fungal conidia, for the transient transformation of Podosphaera fusca (synonym Podosphaera xanthii), the main causal agent of cucurbit powdery mildew.
To introduce DNA into P. xanthii conidia, we applied two square-wave pulses of 1.7 kV for 1 ms with an interval of 5 s. We tested these conditions with several plasmids bearing as selective markers hygromycin B resistance (hph), carbendazim resistance (TUB2) or GFP (gfp) under control of endogenous regulatory elements from Aspergillus nidulans, Neurospora crassa or P. xanthii to drive their expression. An in planta selection procedure using the MBC fungicide carbendazim permitted the propagation of transformants onto zucchini cotyledons and avoided the phytotoxicity associated with hygromycin B.
This is the first report on the transformation of P. xanthii and the transformation of powdery mildew fungi using electroporation. Although the transformants are transient, this represents a feasible method for the genetic manipulation of this important group of plant pathogens.
PMCID: PMC4328038  PMID: 25651833
Electrotransformation; Hygromycin B resistance; MBC resistance; Powdery mildew fungi; β-tubulin
17.  Identification of ferredoxin II as a major calcium binding protein in the nitrogen-fixing symbiotic bacterium Mesorhizobium loti 
BMC Microbiology  2015;15(1):16.
Legumes establish with rhizobial bacteria a nitrogen-fixing symbiosis which is of the utmost importance for both plant nutrition and a sustainable agriculture. Calcium is known to act as a key intracellular messenger in the perception of symbiotic signals by both the host plant and the microbial partner. Regulation of intracellular free Ca2+ concentration, which is a fundamental prerequisite for any Ca2+-based signalling system, is accomplished by complex mechanisms including Ca2+ binding proteins acting as Ca2+ buffers. In this work we investigated the occurrence of Ca2+ binding proteins in Mesorhizobium loti, the specific symbiotic partner of the model legume Lotus japonicus.
A soluble, low molecular weight protein was found to share several biochemical features with the eukaryotic Ca2+-binding proteins calsequestrin and calreticulin, such as Stains-all blue staining on SDS-PAGE, an acidic isoelectric point and a Ca2+-dependent shift of electrophoretic mobility. The protein was purified to homogeneity by an ammonium sulfate precipitation procedure followed by anion-exchange chromatography on DEAE-Cellulose and electroendosmotic preparative electrophoresis. The Ca2+ binding ability of the M. loti protein was demonstrated by 45Ca2+-overlay assays. ESI-Q-TOF MS/MS analyses of the peptides generated after digestion with either trypsin or endoproteinase AspN identified the rhizobial protein as ferredoxin II and confirmed the presence of Ca2+ adducts.
The present data indicate that ferredoxin II is a major Ca2+ binding protein in M. loti that may participate in Ca2+ homeostasis and suggest an evolutionarily ancient origin for protein-based Ca2+ regulatory systems.
Electronic supplementary material
The online version of this article (doi:10.1186/s12866-015-0352-5) contains supplementary material, which is available to authorized users.
PMCID: PMC4322793  PMID: 25648224
Calcium binding proteins; Calcium homeostasis; Ferredoxin II; Mesorhizobium loti; Nitrogen fixation; Rhizobium-legume symbiosis
18.  A new suite of tnaA mutants suggests that Escherichia coli tryptophanase is regulated by intracellular sequestration and by occlusion of its active site 
BMC Microbiology  2015;15(1):14.
The Escherichia coli enzyme tryptophanase (TnaA) converts tryptophan to indole, which triggers physiological changes and regulates interactions between bacteria and their mammalian hosts. Tryptophanase production is induced by external tryptophan, but the activity of TnaA is also regulated by other, more poorly understood mechanisms. For example, the enzyme accumulates as a spherical inclusion (focus) at midcell or at one pole, but how or why this localization occurs is unknown.
TnaA activity is low when the protein forms foci during mid-logarithmic growth but its activity increases as the protein becomes more diffuse, suggesting that foci may represent clusters of inactive (or less active) enzyme. To determine what protein characteristics might mediate these localization effects, we constructed 42 TnaA variants: 6 truncated forms and 36 missense mutants in which different combinations of 83 surface-exposed residues were converted to alanine. A truncated TnaA protein containing only domains D1 and D3 (D1D3) localized to the pole. Mutations affecting the D1D3-to-D1D3 interface did not affect polar localization of D1D3 but did delay assembly of wild type TnaA foci. In contrast, alterations to the D1D3-to-D2 domain interface produced diffuse localization of the D1D3 variant but did not affect the wild type protein. Altering several surface-exposed residues decreased TnaA activity, implying that tetramer assembly may depend on interactions involving these sites. Interestingly, changing any of three amino acids at the base of a loop near the catalytic pocket decreased TnaA activity and caused it to form elongated ovoid foci in vivo, indicating that the alterations affect focus formation and may regulate how frequently tryptophan reaches the active site.
The results suggest that TnaA activity is regulated by subcellular localization and by a loop-associated occlusion of its active site. Equally important, these new TnaA variants are immediately available to the research community and should be useful for investigating how tryptophanase is localized and assembled, how substrate accesses its active site, the functional role of acetylation, and other structural and functional questions.
Electronic supplementary material
The online version of this article (doi:10.1186/s12866-015-0346-3) contains supplementary material, which is available to authorized users.
PMCID: PMC4323232  PMID: 25650045
TnaA; tryptophanase; Cell pole; Protein localization; Enzyme regulation
19.  High temporal resolution of glucosyltransferase dependent and independent effects of Clostridium difficile toxins across multiple cell types 
BMC Microbiology  2015;15(1):7.
Clostridium difficile toxins A and B (TcdA and TcdB), considered to be essential for C. difficile infection, affect the morphology of several cell types with different potencies and timing. However, morphological changes over various time scales are poorly characterized. The toxins’ glucosyltransferase domains are critical to their deleterious effects, and cell responses to glucosyltransferase-independent activities are incompletely understood. By tracking morphological changes of multiple cell types to C. difficile toxins with high temporal resolution, cellular responses to TcdA, TcdB, and a glucosyltransferase-deficient TcdB (gdTcdB) are elucidated.
Human umbilical vein endothelial cells, J774 macrophage-like cells, and four epithelial cell lines (HCT8, T84, CHO, and immortalized mouse cecal epithelial cells) were treated with TcdA, TcdB, gdTcdB. Impedance across cell cultures was measured to track changes in cell morphology. Metrics from impedance data, developed to quantify rapid and long-lasting responses, produced standard curves with wide dynamic ranges that defined cell line sensitivities. Except for T84 cells, all cell lines were most sensitive to TcdB. J774 macrophages stretched and increased in size in response to TcdA and TcdB but not gdTcdB. High concentrations of TcdB and gdTcdB (>10 ng/ml) greatly reduced macrophage viability. In HCT8 cells, gdTcdB did not induce a rapid cytopathic effect, yet it delayed TcdA and TcdB’s rapid effects. gdTcdB did not clearly delay TcdA or TcdB’s toxin-induced effects on macrophages.
Epithelial and endothelial cells have similar responses to toxins yet differ in timing and degree. Relative potencies of TcdA and TcdB in mouse epithelial cells in vitro do not correlate with potencies in vivo. TcdB requires glucosyltransferase activity to cause macrophages to spread, but cell death from high TcdB concentrations is glucosyltransferase-independent. Competition experiments with gdTcdB in epithelial cells confirm common TcdA and TcdB mechanisms, yet different responses of macrophages to TcdA and TcdB suggest different, additional mechanisms or targets in these cells. This first-time, precise quantification of the response of multiple cell lines to TcdA and TcdB provides a comparative framework for delineating the roles of different cell types and toxin-host interactions.
Electronic supplementary material
The online version of this article (doi:10.1186/s12866-015-0361-4) contains supplementary material, which is available to authorized users.
PMCID: PMC4323251  PMID: 25648517
Clostridium difficile; Toxin A; Toxin B; Glucosyltransferase; Epithelial; Endothelial
20.  Genome-wide identification of Bacillus subtilis Zur-binding sites associated with a Zur box expands its known regulatory network 
BMC Microbiology  2015;15(1):13.
The Bacillus subtilis Zur transcription factor recognizes a specific DNA motif, the Zur box, to repress expression of genes in response to zinc availability. Although several Zur-regulated genes are well characterized, a genome-wide mapping of Zur-binding sites is needed to define further the set of genes directly regulated by this protein.
Using chromatin immunoprecipitation coupled with hybridization to DNA tiling arrays (ChIP-on-chip), we reported the identification of 80 inter- and intragenic chromosomal sites bound by Zur. Seven Zur-binding regions constitute the Zur primary regulon while 35 newly identified targets were associated with a predicted Zur box. Using transcriptional fusions an intragenic Zur box was showed to promote a full Zur-mediated repression when placed within a promoter region. In addition, intragenic Zur boxes appeared to mediate a transcriptional cis-repressive effect (4- to 9-fold) but the function of Zur at these sites remains unclear. Zur binding to intragenic Zur boxes could prime an intricate mechanisms of regulation of the transcription elongation, possibly with other transcriptional factors. However, the disruption of zinc homeostasis in Δzur cells likely affects many cellular processes masking direct Zur-dependent effects. Finally, most Zur-binding sites were located near or within genes responsive to disulfide stress. These findings expand the potential Zur regulon and reveal unknown interconnections between zinc and redox homeostasis regulatory networks.
Our findings considerably expand the potential Zur regulon, and reveal a new level of complexity in Zur binding to its targets via a Zur box motif and via a yet unknown mechanism that remains to be characterized.
Electronic supplementary material
The online version of this article (doi:10.1186/s12866-015-0345-4) contains supplementary material, which is available to authorized users.
PMCID: PMC4324032  PMID: 25649915
Zur regulator; B. subtilis; ChIP-on-chip; Zinc homeostasis; Disulfide stress
21.  The immediate global responses of Aliivibrio salmonicida to iron limitations 
BMC Microbiology  2015;15(1):9.
Iron is an essential micronutrient for all living organisms, and virulence and sequestration of iron in pathogenic bacteria are believed to be correlated. As a defence mechanism, potential hosts therefore keep the level of free iron inside the body to a minimum. In general, iron metabolism is well studied for some bacteria (mostly human or animal pathogens). However, this area is still under-investigated for a number of important bacterial pathogens. Aliivibrio salmonicida is a fish pathogen, and previous studies of this bacterium have shown that production of siderophores is temperature regulated and dependent on low iron conditions. In this work we studied the immediate changes in transcription in response to a sudden decrease in iron levels in cultures of A. salmonicida. In addition, we compared our results to studies performed with Vibrio cholerae and Vibrio vulnificus using a pan-genomic approach.
Microarray technology was used to monitor global changes in transcriptional levels. Cultures of A. salmonicida were grown to mid log phase before the iron chelator 2,2’-dipyridyl was added and samples were collected after 15 minutes of growth. Using our statistical cut-off values, we retrieved thirty-two differentially expressed genes where the most up-regulated genes belong to an operon encoding proteins responsible for producing the siderophore bisucaberin. A subsequent pan-transcriptome analysis revealed that nine of the up-regulated genes from our dataset were also up-regulated in datasets from similar experiments using V. cholerae and V. vulnificus, thus indicating that these genes are involved in a shared strategy to mitigate low iron conditions.
The present work highlights the effect of iron limitation on the gene regulatory network of the fish pathogen A. salmonicida, and provides insights into common and unique strategies of Vibrionaceae species to mitigate low iron conditions.
Electronic supplementary material
The online version of this article (doi:10.1186/s12866-015-0342-7) contains supplementary material, which is available to authorized users.
PMCID: PMC4324432  PMID: 25649684
Aliivibrio salmonicida; Iron homeostasis; Ferric uptake regulator; Siderophore; Bisucaberin; Microarray
22.  Impact of the exopolysaccharide layer on biofilms, adhesion and resistance to stress in Lactobacillus johnsonii FI9785 
BMC Microbiology  2015;15(1):8.
The bacterial cell surface is a crucial factor in cell-cell and cell-host interactions. Lactobacillus johnsonii FI9785 produces an exopolysaccharide (EPS) layer whose quantity and composition is altered in mutants that harbour genetic changes in their eps gene clusters. We have assessed the effect of changes in EPS production on cell surface characteristics that may affect the ability of L. johnsonii to colonise the poultry host and exclude pathogens.
Analysis of physicochemical cell surface characteristics reflected by Zeta potential and adhesion to hexadecane showed that an increase in EPS gave a less negative, more hydrophilic surface and reduced autoaggregation. Autoaggregation was significantly higher in mutants that have reduced EPS, indicating that EPS can mask surface structures responsible for cell-cell interactions. EPS also affected biofilm formation, but here the quantity of EPS produced was not the only determinant. A reduction in EPS production increased bacterial adhesion to chicken gut explants, but made the bacteria less able to survive some stresses.
This study showed that manipulation of EPS production in L. johnsonii FI9785 can affect properties which may improve its performance as a competitive exclusion agent, but that positive changes in adhesion may be compromised by a reduction in the ability to survive stress.
Electronic supplementary material
The online version of this article (doi:10.1186/s12866-015-0347-2) contains supplementary material, which is available to authorized users.
PMCID: PMC4326364  PMID: 25648083
Lactobacillus johnsonii; Exopolysaccharides; Biofilm; Adhesion; Cell surface
23.  Dissemination of IncF-type plasmids in multiresistant CTX-M-15-producing Enterobacteriaceae isolates from surgical-site infections in Bangui, Central African Republic 
BMC Microbiology  2015;15(1):15.
Surgical-site infection is the most frequent health care-associated infection in the developing world, with a strikingly higher prevalence than in developed countries We studied the prevalence of resistance to antibiotics in Enterobacteriaceae isolates from surgical-site infections collected in three major tertiary care centres in Bangui, Central African Republic. We also studied the genetic basis for antibiotic resistance and the genetic background of third-generation cephalosporin-resistant (3GC-R) Enterobacteriaceae.
Between April 2011 and April 2012, 195 patients with nosocomial surgical-site infections were consecutively recruited into the study at five surgical departments in three major tertiary care centres. Of the 165 bacterial isolates collected, most were Enterobacteriaceae (102/165, 61.8%). Of these, 65/102 (63.7%) were 3GC-R, which were characterized for resistance gene determinants and genetic background. The blaCTX-M-15 and aac(6′)-Ib-cr genes were detected in all strains, usually associated with qnr genes (98.5%). Escherichia coli, the most commonly recovered species (33/65, 50.8%), occurred in six different sequence types, including the pandemic B2-O25b-ST131 group (12/33, 36.4%). Resistance transfer was studied in one representative strain of the resistance gene content in each repetitive extragenic palindromic and enterobacterial repetitive intergenic consensus sequence-PCR banding pattern. Plasmids were characterized by PCR-based replicon typing and sub-typing schemes. In most isolates (18/27, 66.7%), blaCTX-M-15 genes were found in incompatibility groups F/F31:A4:B1 and F/F36:A4:B1 conjugative plasmids. Horizontal transfer of both plasmids is probably an important mechanism for the spread of blaCTX-M-15 among Enterobacteriaceae species and hospitals. The presence of sets of antibiotic resistance genes in these two plasmids indicates their capacity for gene rearrangement and their evolution into new variants.
Diverse modes are involved in transmission of resistance, plasmid dissemination probably playing a major role.
PMCID: PMC4326526  PMID: 25648151
Antimicrobial resistance; CTX-M-15; IncF; Surgical-site infection; Enterobacteriaceae; Africa
24.  Characterization of the interaction of staphylococcal enterotoxin B with CD1d expressed in human renal proximal tubule epithelial cells 
BMC Microbiology  2015;15(1):12.
Participation of renal cells in the pathogenesis of staphylococcal enterotoxin B (SEB) is critical for late cleansing and sequestration of the antigens facilitated by CD1d mediated antigen sensing and recognition. This is a noted deviation from the typical antigen recognition process that recruits the major histocompatibility complex class II (MHC II) molecules. The immunological importance of CD1d is underscored by its influences on the performances of natural killer T-cells and thereby mediates the innate and adaptive immune systems.
Using diffraction-based dotReady™ immunoassays, the present study showed that SEB directly and specifically conjugated to CD1d. The specificity of the conjugation between SEB and CD1d expressed on human renal proximal tubule epithelial cells (RPTEC) was further established by selective inhibition of CD1d prior to its exposure to SEB. We found that SEB induced the expression of CD1d on the cell surface prompting a rapid conjugation between them. The mRNA transcripts encoding CD1d remained elevated potentially after completing the antigen cleansing process.
Molecular targets associated with the delayed pathogenic response have essential therapeutic values. Particularly in the event of bioterrorism, the caregivers are typically able to intervene much later than the toxic exposures. Given circumstances mandate a paradigm shift from the conventional therapeutic strategy that counts on targeting the host markers responding to the early assault of pathogens. We demonstrated the role of CD1d in the late stage of pathogen recognition and cleansing, and thereby underscored its clinical potential in treating bioweaponizable antigens, such as Staphylococcal enterotoxin B (SEB).
Electronic supplementary material
The online version of this article (doi:10.1186/s12866-015-0344-5) contains supplementary material, which is available to authorized users.
PMCID: PMC4327782  PMID: 25649790
25.  Mesoionic compounds with antifungal activity against Fusarium verticillioides 
BMC Microbiology  2015;15(1):11.
Fungi contaminate the food of humans and animals, are a risk to health, and can cause financial losses. In this work, the antifungal activities of 16 mesoionic compounds (MI 1–16) were evaluated against mycotoxigenic fungi, including Aspergillus spp., Fusarium verticillioides and Penicillium citrinum. Furthermore, the decreased ergosterol in the total lipid content of Fusarium verticillioides was investigated.
F. verticillioides was the most sensitive fungus to the mesoionic compounds. Among the evaluated compounds, MI-11 and MI-16 presented higher antifungal effects against F. verticillioides, with MIC values of 7.8 μg/ml, and MI-2 and MI-3 followed, with MICs of 15.6 μg/ml. The most active compounds were those with heterocyclic ring phenyl groups substituted by electron donor moieties (MI-11 and MI-16). Among some compounds with higher activity (MI-2, MI-11 and MI-16), decreased ergosterol content in the total lipid fraction of F. verticillioides was demonstrated. MI-2 reduced the ergosterol content approximately 40% and 80% at concentrations of 7.8 μg/ml and 15.6 μg/ml, respectively, and MI-11 and MI-16 decreased the content by 30% and 50%, respectively, when at a concentration of 7.8 μg/ml.
These findings indicate that mesoionic compounds have significant antifungal activity against F. verticillioides.
PMCID: PMC4327949  PMID: 25649493
Mycotoxigenic fungi; Mycotoxicology; Mesoionic; Antifungal activity; Ergosterol

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