Mutualistic growth of the sulfate-reducer Desulfovibrio vulgaris Hildenborough with different carbohydrates

Desulfovibrio vulgaris Hildenborough genome presents a phosphotransferase system putatively involved in the transport of carbohydrates. However, utilization of sugars by this sulfate-reducing bacterium has never been reported. Herein, we have observed proliferation of D. vulgaris Hildenborough with some carbohydrates, in mutualism with Stenotrophomonas maltophilia, a non-fermentative, gram-negative gammaproteobacterium, or Microbacterium, a gram-positive actinobacterium. These results suggest the importance of feedback interactions between different heterotrophic bacterial species including the alternative for D. vulgaris of exploiting additional organic resources and novel habitats. Thus, D. vulgaris strongly participates in the mineralization of carbohydrates both in complex natural and artificial systems.     

A direct demonstration of hydrogen cycling by Desulfovibrio vulgaris employing membrane-inlet mass spectrometry

Employing membrane-inlet mass spectrometry, direct evidence is presented that substrate amounts of hydrogen are simultaneously produced and consumed during the metabolism of pyruvate plus sulfate by washed intact cells of Desulfovibrio vulgaris (Hildenborough). The results clearly demonstrate that hydrogen cycling is an important bioenergetic mechanism for this sulfate-reducing bacterium when growing on pyruvate plus sulfate.     

Hybrid Cluster Proteins and Flavodiiron Proteins Afford Protection to Desulfovibrio vulgaris upon Macrophage Infection

Desulfovibrio species are Gram-negative anaerobic sulfate-reducing bacteria that colonize the human gut. Recently, Desulfovibrio spp. have been implicated in gastrointestinal diseases and shown to stimulate the epithelial immune response, leading to increased production of inflammatory cytokines by macrophages. Activated macrophages are key cells of the immune system that impose nitrosative stress during phagocytosis. Hence, we have analyzed the in vitro and in vivo responses of Desulfovibrio vulgaris Hildenborough to nitric oxide (NO) and the role of the hybrid cluster proteins (HCP1 and HCP2) and rubredoxin oxygen oxidoreductases (ROO1 and ROO2) in NO protection. Among the four genes, hcp2 was the gene most highly induced by NO, and the hcp2 transposon mutant exhibited the lowest viability under conditions of NO stress. Studies in murine macrophages revealed that D. vulgaris survives incubation with these phagocytes and triggers NO production at levels similar to those stimulated by the cytokine gamma interferon (IFN-γ). Furthermore, D. vulgaris hcp and roo mutants exhibited reduced viability when incubated with macrophages, revealing that these gene products contribute to the survival of D. vulgaris during macrophage infection.     

Deletion of the rbo Gene Increases the Oxygen Sensitivity of the Sulfate-Reducing Bacterium Desulfovibrio vulgaris Hildenborough

The rbo gene of Desulfovibrio vulgaris Hildenborough encodes rubredoxin oxidoreductase (Rbo), a 14-kDa iron sulfur protein; forms an operon with the gene for rubredoxin; and is preceded by the gene for the oxygen-sensing protein DcrA. We have deleted the rbo gene from D. vulgaris with the sacB mutagenesis procedure developed previously (R. Fu and G. Voordouw, Microbiology 143:1815–1826, 1997). The absence of the rbo-gene in the resulting mutant, D. vulgaris L2, was confirmed by PCR and protein blotting with Rbo-specific polyclonal antibodies. D. vulgaris L2 grows like the wild type under anaerobic conditions. Exposure to air for 24 h caused a 100-fold drop in CFU of L2 relative to the wild type. The lag times of liquid cultures of inocula exposed to air were on average also greater for L2 than for the wild type. These results demonstrate that Rbo, which is not homologous with superoxide dismutase or catalase, acts as an oxygen defense protein in the anaerobic, sulfate-reducing bacterium D. vulgaris Hildenborough and likely also in other sulfate-reducing bacteria and anaerobic archaea in which it has been found.     

Model-based identification of conditionally-essential genes from transposon-insertion sequencing data

The understanding of bacterial gene function has been greatly enhanced by recent advancements in the deep sequencing of microbial genomes. Transposon insertion sequencing methods combines next-generation sequencing techniques with transposon mutagenesis for the exploration of the essentiality of genes under different environmental conditions. We propose a model-based method that uses regularized negative binomial regression to estimate the change in transposon insertions attributable to gene-environment changes in this genetic interaction study without transformations or uniform normalization. An empirical Bayes model for estimating the local false discovery rate combines unique and total count information to test for genes that show a statistically significant change in transposon counts. When applied to RB-TnSeq (randomized barcode transposon sequencing) and Tn-seq (transposon sequencing) libraries made in strains of Caulobacter crescentus using both total and unique count data the model was able to identify a set of conditionally beneficial or conditionally detrimental genes for each target condition that shed light on their functions and roles during various stress conditions.     

Effect of Calcium Cation on Plating Efficiency of the Sulfate-Reducing Bacterium Desulfovibrio vulgaris

The presence of Ca²⁺ (concentration, ca. 0.4 mM) in the growth medium causes cells of Desulfovibrio vulgaris (Hildenborough) to aggregate, leading to a decrease in plating efficiency. When the Ca²⁺ concentration in the medium was reduced 20-fold, cell aggregation did not occur and the plating efficiency increased from an initial value of 34% to a final value of 56%.     

Regulation of the Periplasmic [Fe] Hydrogenase by Ferrous Iron in Desulfovibrio vulgaris (Hildenborough)

The periplasmic [Fe] hydrogenase from the sulfate-reducing bacterium Desulfovibrio vulgaris (Hildenborough) DSM 8303 was found to be regulated by ferrous iron availability. During growth with 5 ppm of iron, the enzyme derepressed and the specific activity increased approximately fourfold, whereas the presence of 100 ppm of ferrous iron repressed the enzyme. The repression-derepression phenomenon with ferrous iron was found to be operative when the cells were cultured under either hydrogen or nitrogen gas. This is the first reported case showing that the hydrogenase enzyme is regulated by iron, and the implications of this finding relative to the corrosion industry are discussed.     

Sequence-Verified Two-Allele Transposon Mutant Library for Pseudomonas aeruginosa PAO1

Mutant hunts using comprehensive sequence-defined libraries make it possible to identify virtually all of the nonessential functions required for different bacterial processes. However, the success of such screening depends on the accuracy of mutant identification in the mutant library used. To provide a high-quality library for Pseudomonas aeruginosa PAO1, we created a sequence-verified collection of 9,437 transposon mutants that provides genome coverage and includes two mutants for most genes. Mutants were cherry-picked from a larger library, colony-purified, and resequenced both individually using Sanger sequencing and in a pool using Tn-seq. About 8% of the insertion assignments were corrected, and in the final library nearly 93% of the transposon locations were confirmed by at least one of the resequencing procedures. The extensive sequence verification and inclusion of more than one mutant for most genes should help minimize missed or erroneous genotype-phenotype assignments in studies using the new library.     

ESSENTIALS: Software for Rapid Analysis of High Throughput Transposon Insertion Sequencing Data

High-throughput analysis of genome-wide random transposon mutant libraries is a powerful tool for (conditional) essential gene discovery. Recently, several next-generation sequencing approaches, e.g. Tn-seq/INseq, HITS and TraDIS, have been developed that accurately map the site of transposon insertions by mutant-specific amplification and sequence readout of DNA flanking the transposon insertions site, assigning a measure of essentiality based on the number of reads per insertion site flanking sequence or per gene. However, analysis of these large and complex datasets is hampered by the lack of an easy to use and automated tool for transposon insertion sequencing data. To fill this gap, we developed ESSENTIALS, an open source, web-based software tool for researchers in the genomics field utilizing transposon insertion sequencing analysis. It accurately predicts (conditionally) essential genes and offers the flexibility of using different sample normalization methods, genomic location bias correction, data preprocessing steps, appropriate statistical tests and various visualizations to examine the results, while requiring only a minimum of input and hands-on work from the researcher. We successfully applied ESSENTIALS to in-house and published Tn-seq, TraDIS and HITS datasets and we show that the various pre- and post-processing steps on the sequence reads and count data with ESSENTIALS considerably improve the sensitivity and specificity of predicted gene essentiality.     

Identification of Listeria monocytogenes Determinants Required for Biofilm Formation

Listeria monocytogenes is a Gram-positive, food-borne pathogen of humans and animals. L. monocytogenes is considered to be a potential public health risk by the U.S. Food and Drug Administration (FDA), as this bacterium can easily contaminate ready-to-eat (RTE) foods and cause an invasive, life-threatening disease (listeriosis). Bacteria can adhere and grow on multiple surfaces and persist within biofilms in food processing plants, providing resistance to sanitizers and other antimicrobial agents. While whole genome sequencing has led to the identification of biofilm synthesis gene clusters in many bacterial species, bioinformatics has not identified the biofilm synthesis genes within the L. monocytogenes genome. To identify genes necessary for L. monocytogenes biofilm formation, we performed a transposon mutagenesis library screen using a recently constructed Himar1 mariner transposon. Approximately 10,000 transposon mutants within L. monocytogenes strain 10403S were screened for biofilm formation in 96-well polyvinyl chloride (PVC) microtiter plates with 70 Himar1 insertion mutants identified that produced significantly less biofilms. DNA sequencing of the transposon insertion sites within the isolated mutants revealed transposon insertions within 38 distinct genetic loci. The identification of mutants bearing insertions within several flagellar motility genes previously known to be required for the initial stages of biofilm formation validated the ability of the mutagenesis screen to identify L. monocytogenes biofilm-defective mutants. Two newly identified genetic loci, dltABCD and phoPR, were selected for deletion analysis and both ΔdltABCD and ΔphoPR bacterial strains displayed biofilm formation defects in the PVC microtiter plate assay, confirming these loci contribute to biofilm formation by L. monocytogenes.     

Rapid selective sweep of pre-existing polymorphisms and slow fixation of new mutations in experimental evolution of Desulfovibrio vulgaris

To investigate the genetic basis of microbial evolutionary adaptation to salt (NaCl) stress, populations of Desulfovibrio vulgaris Hildenborough (DvH), a sulfate-reducing bacterium important for the biogeochemical cycling of sulfur, carbon and nitrogen, and potentially the bioremediation of toxic heavy metals and radionuclides, were propagated under salt stress or non-stress conditions for 1200 generations. Whole-genome sequencing revealed 11 mutations in salt stress-evolved clone ES9-11 and 14 mutations in non-stress-evolved clone EC3-10. Whole-population sequencing data suggested the rapid selective sweep of the pre-existing polymorphisms under salt stress within the first 100 generations and the slow fixation of new mutations. Population genotyping data demonstrated that the rapid selective sweep of pre-existing polymorphisms was common in salt stress-evolved populations. In contrast, the selection of pre-existing polymorphisms was largely random in EC populations. Consistently, at 100 generations, stress-evolved population ES9 showed improved salt tolerance, namely increased growth rate (2.0-fold), higher biomass yield (1.8-fold) and shorter lag phase (0.7-fold) under higher salinity conditions. The beneficial nature of several mutations was confirmed by site-directed mutagenesis. All four tested mutations contributed to the shortened lag phases under higher salinity condition. In particular, compared with the salt tolerance improvement in ES9-11, a mutation in a histidine kinase protein gene lytS contributed 27% of the growth rate increase and 23% of the biomass yield increase while a mutation in hypothetical gene DVU2472 contributed 24% of the biomass yield increase. Our results suggested that a few beneficial mutations could lead to dramatic improvements in salt tolerance.     

Sequence and activity of an endogenous promoter of the assimilatory sulfite reductase gene from Desulfovibrio vulgaris (Hildenborough)

Summary The 5-flanking region of the assimilatory-type sulfite reductase-encoding gene from the anaerobic bacterium, Desulfovibrio vulgaris (Hildenborough), has been isolated and sequenced. The promoter element has been identified and compared with the consensus sequence for Escherichia coli and the sequences of promoter elements identified in other Desulfovibrio strains. The utility of such promoter sequences in E. coli-based expression systems is discussed.     

DsrC is involved in fermentative growth and interacts directly with the FlxABCD–HdrABC complex in Desulfovibrio vulgaris Hildenborough

DsrC is a key protein in dissimilatory sulfur metabolism, where it works as co-substrate of the dissimilatory sulfite reductase DsrAB. DsrC has two conserved cysteines in a C-terminal arm that are converted to a trisulfide upon reduction of sulfite. In sulfate-reducing bacteria, DsrC is essential and previous works suggested additional functions beyond sulfite reduction. Here, we studied whether DsrC also plays a role during fermentative growth of Desulfovibrio vulgaris Hildenborough, by studying two strains where the functionality of DsrC is impaired by a lower level of expression (IPFG07) and additionally by the absence of one conserved Cys (IPFG09). Growth studies coupled with metabolite and proteomic analyses reveal that fermentation leads to lower levels of DsrC, but impairment of its function results in reduced growth by fermentation and a shift towards more fermentative metabolism during sulfate respiration. In both respiratory and fermentative conditions, there is increased abundance of the FlxABCD–HdrABC complex and Adh alcohol dehydrogenase in IPFG09 versus the wild type, which is reflected in higher production of ethanol. Pull-down experiments confirmed a direct interaction between DsrC and the FlxABCD–HdrABC complex, through the HdrB subunit. Dissimilatory sulfur metabolism, where sulfur compounds are used for energy generation, is a key process in the ecology of anoxic environments, and is more widespread among bacteria than previously believed. Two central proteins for this type of metabolism are DsrAB dissimilatory sulfite reductase and its co-substrate DsrC. Using physiological, proteomic and biochemical studies of Desulfovibrio vulgaris Hildenborough and mutants affected in DsrC functionality, we show that DsrC is also relevant for fermentative growth of this model organism and that it interacts directly with the soluble FlxABCD-HdrABC complex that links the NAD(H) pool with dissimilatory sulfite reduction.     

mariner-Based Transposon Mutagenesis of Rickettsia prowazekii

Rickettsia prowazekii, the causative agent of epidemic typhus, is an obligate intracellular bacterium that grows directly within the cytoplasm of its host cell, unbounded by a vacuolar membrane. The obligate intracytoplasmic nature of rickettsial growth places severe restrictions on the genetic analysis of this distinctive human pathogen. In order to expand the repertoire of genetic tools available for the study of this pathogen, we have employed the versatile mariner-based, Himar1 transposon system to generate insertional mutants of R. prowazekii. A transposon containing the R. prowazekii arr-2 rifampin resistance gene and a gene coding for a green fluorescent protein (GFP_UV) was constructed and placed on a plasmid expressing the Himar1 transposase. Electroporation of this plasmid into R. prowazekii resulted in numerous transpositions into the rickettsial genome. Transposon insertion sites were identified by rescue cloning, followed by DNA sequencing. Random transpositions integrating at TA sites in both gene coding and intergenic regions were identified. Individual rickettsial clones were isolated by the limiting-dilution technique. Using both fixed and live-cell techniques, R. prowazekii transformants expressing GFP_UV were easily visible by fluorescence microscopy. Thus, a mariner-based system provides an additional mechanism for generating rickettsial mutants that can be screened using GFP_UV fluorescence.     

Quantitative target display: a method to screen yeast mutants conferring quantitative phenotypes by ‘mutant DNA fingerprints’

Whole genome sequencing of several microbes has revealed thousands of genes of unknown function. A large proportion of these genes seem to confer subtle quantitative phenotypes or phenotypes that do not have a plate screen. We report a novel method to monitor such phenotypes, where the fitness of mutants is assessed in mixed cultures under competitive growth conditions, and the abundance of any individual mutant in the pool is followed by means of its unique feature, namely the mutation itself. A mixed population of yeast mutants, obtained through transposon mutagenesis, was subjected to selection. The DNA regions (targets) flanking the transposon, until nearby restriction sites, are then quantitatively amplified by means of a ligation-mediated PCR method, using transposon-specific and adapter-specific primers. The amplified PCR products correspond to mutated regions of the genome and serve as ‘mutant DNA fingerprints’ that can be displayed on a sequencing gel. The relative intensity of the amplified DNA fragments before and after selection match with the relative abundance of corresponding mutants, thereby revealing the fate of the mutants during selection. Using this method we demonstrate that UBI4, YDJ1 and HSP26 are essential for stress tolerance of yeast during ethanol production. We anticipate that this method will be useful for functional analysis of genes of any microbe amenable to insertional mutagenesis.     

Genome scanning for conditionally essential genes in Salmonella enterica Serotype Typhimurium

As more whole-genome sequences become available, there is an increasing demand for high-throughput methods that link genes to phenotypes, facilitating discovery of new gene functions. In this study, we describe a new version of the Tn-seq method involving a modified EZ:Tn5 transposon for genome-wide and quantitative mapping of all insertions in a complex mutant library utilizing massively parallel Illumina sequencing. This Tn-seq method was applied to a genome-saturating Salmonella enterica serotype Typhimurium mutant library recovered from selection under 3 different in vitro growth conditions (diluted Luria-Bertani [LB] medium, LB medium plus bile acid, and LB medium at 42°C), mimicking some aspects of host stressors. We identified an overlapping set of 105 protein-coding genes in S. Typhimurium that are conditionally essential under at least one of the above selective conditions. Competition assays using 4 deletion mutants (pyrD, glnL, recD, and STM14_5307) confirmed the phenotypes predicted by Tn-seq data, validating the utility of this approach in discovering new gene functions. With continuously increasing sequencing capacity of next generation sequencing technologies, this robust Tn-seq method will aid in revealing unexplored genetic determinants and the underlying mechanisms of various biological processes in Salmonella and the other approximately 70 bacterial species for which EZ:Tn5 mutagenesis has been established.