BMC Microbiology

Coxiella burnetii causes Q fever in humans and Coxiellosis in animals; symptoms range from general malaise to fever, pneumonia, endocarditis and death. Livestock are a significant source of human infection as they shed C. burnetii cells in birth tissues, milk, urine and feces. Although prevalence of C. burnetii is high, few Q fever cases are reported in the U.S. and we have a limited understanding of their connectedness due to difficulties in genotyping. Here, we develop canonical SNP genotyping assays to evaluate spatial and temporal relationships among C. burnetii environmental samples and compare them across studies. Given the genotypic diversity of historical collections, we hypothesized that the current enzootic of Coxiellosis is caused by multiple circulating genotypes. We collected A) 23 milk samples from a single bovine herd, B) 134 commercial bovine and caprine milk samples from across the U.S., and C) 400 bovine and caprine samples from six milk processing plants over three years. We detected C. burnetii DNA in 96% of samples with no variance over time. We genotyped 88.5% of positive samples; bovine milk contained only a single genotype (ST20) and caprine milk was dominated by a second type (mostly ST8). The high prevalence and lack of genotypic diversity is consistent with a model of rapid spread and persistence. The segregation of genotypes between host species is indicative of species-specific adaptations or dissemination barriers and may offer insights into the relative lack of human cases and characterizing genotypes.

We have recently shown that undomesticated strains of Bacillus subtilis can extensively colonize the surfaces of rich, semi-solid media, by a flagellum-independent mechanism and suggested that sliding motility is responsible for surface migration. Here we have used a flagella-less hag null mutant to examine and confirm sliding motility. Using a defined semi-solid medium we determined that a B. subtilis hag mutant colonized the surface in two stages, first as tendril-like clusters of cells followed by a profuse pellicle-like film. We determined the levels of macro- and micro-nutrients required for the tendril-to-film transition. Sufficient levels of each of the macronutrients, glycerol, Na-glutamate, and Na-phosphate, and inorganic nutrients, K+, Mg2+, Fe2+ and Mn2+, were required for robust film formation. The K+ requirement was quantified in more detail, and the thresholds for complete tendril coverage (50 μM KCl) or film coverage (2–3 mM KCl) were determined. In addition, disruption of the genes for the higher affinity K+ transporter (KtrAB), but not the lower affinity K+ transporter (KtrCD), strongly inhibited the formation of both tendrils and films, and could be partially overcome by high levels of KCl. Examination of hag tendrils by confocal scanning laser microscopy revealed that tendrils are multicellular structures, but that the cells are not as highly organized as cells in wild-type B. subtilis pellicles. These results suggest that B. subtilis can use sliding motility to colonize surfaces, using a tendril-like growth mode when various macronutrients or micronutrients are limiting. If nutrients are balanced and sufficient, the surfaces between tendrils can be colonized by robust surface films. Sliding motility may represent a strategy for nutrient-deprived cells to colonize surfaces in natural environments, such as plant roots, and the media described here may be useful in investigations of this growth phenotype.

Abstract Background Much of the Plasmodium falciparum genome encodes hypothetical proteins with limited homology to other organisms. A lack of robust tools for genetic manipulation of the parasite limits functional analysis of these hypothetical proteins and other aspects of the Plasmodium genome. Transposon mutagenesis has been used widely to identify gene functions in many organisms and would be extremely valuable for functional analysis of the Plasmodium genome. Results In this study, we investigated the lepidopteran transposon, piggyBac, as a molecular genetic tool for functional characterization of the Plasmodium falciparum genome. Through multiple transfections, we generated 177 unique P. falciparum mutant clones with mostly single piggyBac insertions in their genomes. Analysis of piggyBac insertion sites revealed random insertions into the P. falciparum genome, in regards to gene expression in parasite life cycle stages and functional categories. We further explored the possibility of forward genetic studies in P. falciparum with a phenotypic screen for attenuated growth, which identified several parasite genes and pathways critical for intra-erythrocytic development. Conclusion Our results clearly demonstrate that piggyBac is a novel, indispensable tool for forward functional genomics in P. falciparum that will help better understand parasite biology and accelerate drug and vaccine development.

In bioethanol production, the main by-product, 5-hydroxymethylfurfural (HMF), significantly hinders microbial fermentation. Therefore, it is crucial to explore genes related to HMF tolerance in Saccharomyces cerevisiae for enhancing the tolerance of ethanol fermentation strains. A comprehensive analysis was conducted using genome-wide deletion library scanning and SGAtools, resulting in the identification of 294 genes associated with HMF tolerance in S. cerevisiae. Further KEGG and GO enrichment analysis revealed the involvement of genes OCA1 and SIW14 in the protein phosphorylation pathway, underscoring their role in HMF tolerance. Spot test validation and subcellular structure observation demonstrated that, following a 3-h treatment with 60 mM HMF, the SIW14 gene knockout strain exhibited a 12.68% increase in cells with abnormal endoplasmic reticulum (ER) and a 22.41% increase in the accumulation of reactive oxygen species compared to the BY4741 strain. These findings indicate that the SIW14 gene contributes to the protection of the ER structure within the cell and facilitates the clearance of reactive oxygen species, thereby confirming its significance as a key gene for HMF tolerance in S. cerevisiae.

Ralstonia eutropha H16 is well known to produce polyhydroxyalkanoates (PHAs), which are potential bio-based biodegradable plastics, in an efficient manner as an energy storage material under unbalanced growth conditions. To obtain further knowledge of PHA biosynthesis, this study performed a quantitative transcriptome analysis based on deep sequencing of the complementary DNA generated from the RNA (RNA-seq) of R. eutropha H16. Total RNAs were extracted from R. eutropha cells in growth, PHA production, and stationary phases on fructose. rRNAs in the preparation were removed by repeated treatments with magnetic beads specific to bacterial rRNAs, and then the 36 bp sequences were determined using an Illumina high-throughput sequencer. The RNA-seq results indicated the induction of gene expression for transcription, translation, cell division, peptidoglycan biosynthesis, pilus and flagella assembly, energy conservation, and fatty acid biosynthesis in the growth phase; and the repression trends of genes involved in central metabolisms in the PHA production phase. Interestingly, the transcription of genes for Calvin-Benson-Bassham (CBB) cycle and several genes for β-oxidation were significantly induced in the PHA production phase even when the cells were grown on fructose. Moreover, incorporation of 13C was observed in poly(3-hydroxybutyrate) synthesized by R. eutropha H16 from fructose in the presence of NaH13CO3, and further gene deletion analyses revealed that both of the two ribulose 1,5-bisphosphate carboxylase (Rubiscos) in CBB cycle were actually functional in CO2 fixation under the heterotrophic condition. The results revealed the phase-dependent transcriptomic changes and a CO2 fixation capability under heterotrophic conditions by PHA-producing R. eutropha.

Bacterial and Archaeal communities have a complex, symbiotic role in crude oil bioremediation. Their biosurfactants and degradation enzymes have been in the spotlight, mainly due to the awareness of ecosystem pollution caused by crude oil accidents and their use. Initially, the scientific community studied the role of individual microbial species by characterizing and optimizing their biosurfactant and oil degradation genes, studying their individual distribution. However, with the advances in genomics, in particular with the use of New-Generation-Sequencing and Metagenomics, it is now possible to have a macro view of the complex pathways related to the symbiotic degradation of hydrocarbons and surfactant production. It is now possible, although more challenging, to obtain the DNA information of an entire microbial community before automatically characterizing it. By characterizing and understanding the interconnected role of microorganisms and the role of degradation and biosurfactant genes in an ecosystem, it becomes possible to develop new biotechnological approaches for bioremediation use. This paper analyzes 46 different metagenome samples, spanning 20 biomes from different geographies obtained from different research projects. A metagenomics bioinformatics pipeline, focused on the biodegradation and biosurfactant-production pathways, genes and organisms, was applied. Our main results show that: (1) surfactation and degradation are correlated events, and therefore should be studied together; (2) terrestrial biomes present more degradation genes, especially cyclic compounds, and less surfactation genes, when compared to water biomes; and (3) latitude has a significant influence on the diversity of genes involved in biodegradation and biosurfactant production. This suggests that microbiomes found near the equator are richer in genes that have a role in these processes and thus have a higher biotechnological potential. In this work we have focused on the biogeographical distribution of hydrocarbon degrading and biosurfactant producing genes. Our principle results can be seen as an important step forward in the application of bioremediation techniques, by considering the biostimulation, optimization or manipulation of a starting microbial consortia from the areas with higher degradation and biosurfactant producing genetic diversity.

We have recently found that Pseudomonas putida deficient in ColRS two-component system is sensitive to phenol and displays a serious defect on solid glucose medium where subpopulation of bacteria lyses. The latter phenotype is significantly enhanced by the presence of phenol in growth medium. Here, we focused on identification of factors affecting phenol tolerance of the colR-deficient P. putida. By using transposon mutagenesis approach we identified a set of phenol-tolerant derivatives of colR-deficient strain. Surprisingly, half of independent phenol tolerant clones possessed miniTn5 insertion in the ttgABC operon. However, though inactivation of TtgABC efflux pump significantly enhanced phenol tolerance, it did not affect phenol-enhanced autolysis of the colR mutant on glucose medium indicating that phenol- and glucose-caused stresses experienced by the colR-deficient P. putida are not coupled. Inactivation of TtgABC pump significantly increased the phenol tolerance of the wild-type P. putida as well. Comparison of phenol tolerance of growing versus starving bacteria revealed that both ColRS and TtgABC systems affect phenol tolerance only under growth conditions and not under starvation. Flow cytometry analysis showed that phenol strongly inhibited cell division and to some extent also caused cell membrane permeabilization to propidium iodide. Single cell analysis of populations of the ttgC- and colRttgC- deficient strains revealed that their membrane permeabilization by phenol resembles that of the wild-type and the colR mutant, respectively. However, cell division of P. putida with inactivated TtgABC pump seemed to be less sensitive to phenol than that of the parental strain. At the same time, cell division appeared to be more inhibited in the colR-mutant strain than in the wild-type P. putida. ColRS signal system and TtgABC efflux pump are involved in the phenol tolerance of P. putida. However, as they affect phenol tolerance of growing bacteria only, this indicates that they participate in the regulation of processes which are active during the growth and/or cell division. Single cell analysis data indicated that the cell division step of cell cycle is particularly sensitive to the toxic effect of phenol and its inhibition can be considered as an adaptive response under conditions of phenol stress.

Exopolysaccharides (EPSs) produced by lactic acid bacteria are important for the texture of fermented foods and have received a great deal of interest recently. However, the low production levels of EPSs in combination with the complex media used for growth of the bacteria have caused problems in the accurate analysis of the EPS. The purpose of this study was to find a growth medium for physiological studies of the lactic acid bacterium Streptococcus thermophilus, and to develop a simple method for qualitative and quantitative analysis of EPSs produced in this medium. A semi-defined polysaccharide medium was developed and evaluated on six strains of Streptococcus thermophilus. The EPSs were analysed using a novel protocol incorporating ultracentrifugation for the removal of interfering sugars, hydrolysis and analysis of the monomer composition by High Performance Anion-Exchange Chromatography with pulsed amperometric detection. The medium and analysis method allowed accurate quantification and monomer analysis of 0.5 ml samples of EPSs from tube cultures. The presented medium should be useful for physiological studies of S. thermophilus, and, in combination with the method of analysis of EPS, will allow downscaling of physiological studies and screening for EPSs.

Leptospira interrogans is a spirochaete responsible for leptospirosis in mammals. The molecular mechanisms of the Leptospira virulence remain mostly unknown. Recently, it has been demonstrated that L. interrogans ClpB (ClpBLi) is essential for bacterial survival under stressful conditions and also during infection. The aim of this study was to provide further insight into the role of ClpB in L. interrogans and answer the question whether ClpBLi as a potential virulence factor may be a target of the humoral immune response during leptospiral infections in mammals. ClpBLi consists of 860 amino acid residues with a predicted molecular mass of 96.3 kDa and shows multi-domain organization similar to that of the well-characterized ClpB from Escherichia coli. The amino acid sequence identity between ClpBLi and E. coli ClpB is 52 %. The coding sequence of the clpB Li gene was cloned and expressed in E. coli BL21(DE3) strain. Immunoreactivity of the recombinant ClpBLi protein was assessed with the sera collected from Leptospira-infected animals and uninfected healthy controls. Western blotting and ELISA analysis demonstrated that ClpBLi activates the host immune system, as evidenced by an increased level of antibodies against ClpBLi in the sera from infected animals, as compared to the control group. Additionally, ClpBLi was found in kidney tissues of Leptospira-infected hamsters. ClpBLi is both synthesized and immunogenic during the infectious process, further supporting its involvement in the pathogenicity of Leptospira. In addition, the immunological properties of ClpBLi point to its potential value as a diagnostic antigen for the detection of leptospirosis.