Mesorhizobium alhagi CCNWXJ12-2 is a α-proteobacterium which could be able to fix nitrogen in the nodules formed with Alhagi sparsifolia in northwest of China. Desiccation and high salinity are the two major environmental problems faced by M. alhagi CCNWXJ12-2. In order to identify genes involved in salt-stress adaption, a global transcriptional analysis of M. alhagi CCNWXJ12-2 growing under salt-free and high salt conditions was carried out. The next generation sequencing technology, RNA-Seq, was used to obtain the transcription profiles.
We have compared the transcriptome of M. alhagi growing in TY medium under high salt conditions (0.4 M NaCl) with salt free conditions as a control. A total of 1,849 differentially expressed genes (fold change ≧ 2) were identified and 933 genes were downregulated while 916 genes were upregulated under high salt condition. Except for the upregulation of some genes proven to be involved in salt resistance, we found that the expression levels of protein secretion systems were changed under high salt condition and the expression levels of some heat shock proteins were reduced by salt stress. Notably, a gene encoding YadA domain-containing protein (yadA), a gene encoding trimethylamine methyltransferase (mttB) and a gene encoding formate--tetrahydrofolate ligase (fhs) were highly upregulated. Growth analysis of the three gene knockout mutants under salt stress demonstrated that yadA was involved in salt resistance while the other two were not.
To our knowledge, this is the first report about transcriptome analysis of a rhizobia using RNA-Seq to elucidate the salt resistance mechanism. Our results showed the complex mechanism of bacterial adaption to salt stress and it was a systematic work for bacteria to cope with the high salinity environmental problems. Therefore, these results could be helpful for further investigation of the bacterial salt resistance mechanism.
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Salt stress; RNA-Seq; Secretion system; Chaperones; Mesorhizobium alhagi
Forestomach fermentation in Australian marsupials such as wallabies and kangaroos, though analogous to rumen fermentation, results in lower methane emissions. Insights into hydrogenotrophy in these systems could help in devising strategies to reduce ruminal methanogenesis. Reductive acetogenesis may be a significant hydrogen sink in these systems and previous molecular analyses have revealed a novel diversity of putative acetogens in the tammar wallaby forestomach.
Methanogen-inhibited enrichment cultures prepared from tammar wallaby forestomach contents consumed hydrogen and produced primarily acetate. Functional gene (formyltetrahydrofolate synthetase and acetyl-CoA synthase) analyses revealed a restricted diversity of Clostridiales species as the putative acetogens in the cultures. A new acetogen (growth on H2/CO2 with acetate as primary end product) designated isolate TWA4, was obtained from the cultures. Isolate TWA4 classified within the Lachnospiraceae and demonstrated >97% rrs identity to previously isolated kangaroo acetogens. Isolate TWA4 was a potent hydrogenotroph and demonstrated excellent mixotrophic growth (concomitant consumption of hydrogen during heterotrophic growth) with glycerol. Mixotrophic growth of isolate TWA4 on glycerol resulted in increased cell densities and acetate production compared to autotrophic growth. Co-cultures with an autotrophic methanogen Methanobrevibacter smithii revealed that isolate TWA4 performed reductive acetogenesis under high hydrogen concentration (>5 mM), but not at low concentrations. Under heterotrophic growth conditions, isolate TWA4 did not significantly stimulate methanogenesis in a co-culture with M. smithii contrary to the expectation for organisms growing fermentatively.
The unique properties of tammar wallaby acetogens might be contributing factors to reduced methanogen numbers and methane emissions from tammar wallaby forestomach fermentation, compared to ruminal fermentation. The macropod forestomach may be a useful source of acetogens for future strategies to reduce methane emissions from ruminants, particularly if these strategies also include some level of methane suppression and/or acetogen stimulation, for example by harnessing mixotrophic growth capabilities
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Acetogen; Acetogenesis; Tammar wallaby; Rumen; Methanogenesis
Green and blue mold decay, caused by Penicillium digitatum and P. italicum, respectively, are important postharvest diseases of citrus. Biocontrol by microbes is an alternative to synthetic fungicide application. In this study, the antagonistic yeast strain Kloeckera apiculata 34–9 was used to investigate the action mechanisms involved in the biocontrol of postharvest diseases.
An antifungal substance, 2-phenylethanol (PEA), was isolated from K. apiculata and demonstrated to have antimicrobial activity against selected phytopathogenic fungi. Experiments on P. italicum cells identified the mitochondria and the nucleus as particularly sensitive to inhibition. Regulation of P. italicum gene expression was investigated using RNA-Seq. PEA up-regulated genes involved with the peroxisome, regulation of autophagy, phosphatidylinositol signaling system, protein processing in endoplasmic reticulum, fatty acid metabolism, and inhibited ribosome, RNA polymerase, DNA replication, amino acid biosynthesis, aminoacyl-tRNA biosynthesis and cell cycle. Inhibitory responses revealed by RNA-Seq suggest that PEA might compete for attachment on the active site of phenylalanyl-tRNA synthetase (PheRS).
This study provided new insight on the mode of action of biocontrol yeast agents in controlling postharvest pathogenic fungi.
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Biological control; Penicillium; Kloeckera apiculata; Antifungal compound; 2-phenylethanol (PEA); Postharvest
Acquisition of exogenous genetic material is a key event in bacterial speciation. It seems reasonable to assume that recombination of the incoming DNA into genome would be more efficient with higher levels of relatedness between the DNA donor and recipient. If so, bacterial speciation would be a smooth process, leading to a continuous spectrum of genomic divergence of bacteria, which, however, is not the case as shown by recent findings. The goal of this study was todetermine if DNA transfer efficiency is correlated with the levels of sequence identity.
To compare the relative efficiency of exogenous DNA acquisition among closely related bacteria, we carried out phage-mediated transduction and plasmid-mediated transformation in representative Salmonella strains with different levels of relatedness. We found that the efficiency was remarkably variable even among genetically almost identical bacteria. Although there was a general tendency that more closely related DNA donor-recipient pairs had higher transduction efficiency, transformation efficiency exhibited over a thousand times difference among the closely related Salmonella strains.
DNA acquisition efficiency is greatly variable among bacteria that have as high as over 99% identical genetic background, suggesting that bacterial speciation involves highly complex processes affected not only by whether beneficial exogenous DNA may exist in the environment but also the “readiness” of the bacteria to accept it.
Bacterial speciation; Homologous recombination; Salmonella; Transduction; Transformation
The emergence and wide distribution of the transferable gene for linezolid resistance, cfr, in staphylococci of human and animal origins is of great concern as it poses a serious threat to the public health. In the present study, we investigated the emergence and presence of the multiresistance gene, cfr, in retail meat sourced from supermarkets and free markets of Guangzhou, China.
A total of 118 pork and chicken samples, collected from Guangzhou markets, were screened by PCR for cfr. Twenty-two Staphylococcus isolates obtained from 12 pork and 10 chicken samples harbored cfr. The 22 cfr-positive staphylococci isolates, including Staphylococcus equorum (n = 8), Staphylococcus simulans (n = 7), Staphylococcus cohnii (n = 4), and Staphylococcus sciuri (n = 3), exhibited 17 major SmaI pulsed-field gel electrophoresis (PFGE) patterns. In 14 isolates, cfr was located on the plasmids. Sequence analysis revealed that the genetic structures (including ΔtnpA of Tn558, IS21-558, ΔtnpB, and tnpC of Tn558, orf138, fexA) of cfr in plasmid pHNTLD18 of a S. sciuri strain and in the plasmid pHNLKJC2 (including rep, Δpre/mob, cfr, pre/mob and partial ermC) of a S. equorum strain were identical or similar to the corresponding regions of some plasmids in staphylococcal species of animal and human origins.
To the best of our knowledge, this is the first study to report the presence of the multiresistance gene, cfr, in animal meat. A high occurrence of cfr was observed in the tested retail meat samples. Thus, it is important to monitor the presence of cfr in animal foods in China.
Plasmids; Linezolid; Staphylococcus spp; Food safety; Resistance epidemiology
Latent tuberculosis infection (LTBI) relies on a homeostasis of macrophages and Mycobacterium tuberculosis (Mtb). The small heat shock protein, Mtb Hsp16.3 (also known as latency-associated antigen), plays an important role in Mtb persistence within macrophages. However, the mechanism of LTBI remains elusive. The aim of this study was to delineate LTBI-related miRNA expression in U937 macrophages expressing Mtb Hsp16.3 protein. U937 macrophages were infected with an integrase-deficient Lentivirus vector to transiently express Mtb Hsp16.3, and green fluorescent protein (GFP) as a control. We used a microRNA (miRNA) microarray chip containing more than 1000 probes to identify the significant differentially expressed miRNAs in the infected U937 cells, and employed real-time quantitative polymerase chain reaction (qRT-PCR) for validation. Furthermore, we confirmed these candidate LTBI-related miRNAs in peripheral blood mononuclear cells from subjects with LTBI and in healthy control individuals. Functional annotation prediction of miRNA target genes and pathway enrichment analyses were used to explore the putative links between these miRNAs and LTBI.
Analysis of the miRNA expression profile identified 149 miRNAs that were differentially expressed in U937 macrophages expressing Mtb Hsp16.3 compared with the control expressing GFP. The expression level of seven miRNAs (miR-424-5p, miR-493-5p, miR-296-5p, miR-27b-3p, miR-377-5p, miR-3680-5p, miR-191-5p) were validated by qRT-PCR. The expression level of four miRNAs (miR-424-5p, miR-27b-3p, miR-377-5p, miR-3680-5p) in the peripheral blood mononuclear cells samples from LTBI and healthy participants reflected the altered patterns observed in the microarray profile. The bioinformatic analyses suggest that the miRNAs may regulate Mtb latent infection by affecting the development of macrophage cells.
The results suggest that miRNA expression may play a considerable role in the pathogenesis of LTBI, and this would increase our understanding of the molecular basis of Hsp16.3-facilitated Mtb survival in macrophages.
microRNAs; Macrophages; Mycobacterium; Tuberculosis; Small heat shock protein; Latent tuberculosis infection
For a long time, Enterococcus faecium was considered a harmless commensal of the mammalian gastrointestinal (GI) tract and was used as a probiotic in fermented foods. In recent decades, E. faecium has been recognised as an opportunistic pathogen that causes diseases such as neonatal meningitis, urinary tract infections, bacteremia, bacterial endocarditis and diverticulitis. E. faecium could be taken into space with astronauts and exposed to the space environment. Thus, it is necessary to observe the phenotypic and molecular changes of E. faecium after spaceflight.
An E. faecium mutant with biochemical features that are different from those of the wild-type strain was obtained from subculture after flight on the SHENZHOU-8 spacecraft. To understand the underlying mechanism causing these changes, the whole genomes of both the mutant and the WT strains were sequenced using Illumina technology. The genomic comparison revealed that dprA, a recombination-mediator gene, and arpU, a gene associated with cell wall growth, were mutated. Comparative transcriptomic and proteomic analyses showed that differentially expressed genes or proteins were involved with replication, recombination, repair, cell wall biogenesis, glycometabolism, lipid metabolism, amino acid metabolism, predicted general function and energy production/conversion.
This study analysed the comprehensive genomic, transcriptomic and proteomic changes of an E. faecium mutant from subcultures that were loaded on the SHENZHOU-8 spacecraft. The implications of these gene mutations and expression changes and their underlying mechanisms should be investigated in the future. We hope that the current exploration of multiple “-omics” analyses of this E. faecium mutant will provide clues for future studies on this opportunistic pathogen.
E. faecium; Genome; Transcriptome; Proteome; Multi-omics
Klebsiella pneumoniae displaying the hypermucoviscosity (HV) phenotype are considered more virulent than HV-negative strains. Nevertheless, the emergence of tissue-abscesses-associated HV-negative isolates motivated us to re-evaluate the role of HV-phenotype.
Instead of genetically manipulating the HV-phenotype of K. pneumoniae, we selected two clinically isolated K1 strains, 1112 (HV-positive) and 1084 (HV-negative), to avoid possible interference from defects in the capsule. These well-encapsulated strains with similar genetic backgrounds were used for comparative analysis of bacterial virulence in a pneumoniae or a liver abscess model generated in either naïve or diabetic mice. In the pneumonia model, the HV-positive strain 1112 proliferated to higher loads in the lungs and blood of naïve mice, but was less prone to disseminate into the blood of diabetic mice compared to the HV-negative strain 1084. In the liver abscess model, 1084 was as potent as 1112 in inducing liver abscesses in both the naïve and diabetic mice. The 1084-infected diabetic mice were more inclined to develop bacteremia and had a higher mortality rate than those infected by 1112. A mini-Tn5 mutant of 1112, isolated due to its loss of HV-phenotype, was avirulent to mice.
These results indicate that the HV-phenotype is required for the virulence of the clinically isolated HV-positive strain 1112. The superior ability of the HV-negative stain 1084 over 1112 to cause bacteremia in diabetic mice suggests that factors other than the HV phenotype were required for the systemic dissemination of K. pneumoniae in an immunocompromised setting.
The effects of enterolignans, e.g., enterodiol (END) and particularly its oxidation product, enterolactone (ENL), on prevention of hormone-dependent diseases, such as osteoporosis, cardiovascular diseases, hyperlipemia, breast cancer, colon cancer, prostate cancer and menopausal syndrome, have attracted much attention. To date, the main way to obtain END and ENL is chemical synthesis, which is expensive and inevitably leads to environmental pollution. To explore a more economic and eco-friendly production method, we explored biotransformation of enterolignans from precursors contained in defatted flaxseeds by human intestinal bacteria.
We cultured fecal specimens from healthy young adults in media containing defatted flaxseeds and detected END from the culture supernatant. Following selection through successive subcultures of the fecal microbiota with defatted flaxseeds as the only carbon source, we obtained a bacterial consortium, designated as END-49, which contained the smallest number of bacterial types still capable of metabolizing defatted flaxseeds to produce END. Based on analysis with pulsed field gel electrophoresis, END-49 was found to consist of five genomically distinct bacterial lineages, designated Group I-V, with Group I strains dominating the culture. None of the individual Group I-V strains produced END, demonstrating that the biotransformation of substrates in defatted flaxseeds into END is a joint work by different members of the END-49 bacterial consortium. Interestingly, Group I strains produced secoisolariciresinol, an important intermediate of END production; 16S rRNA analysis of one Group I strain established its close relatedness with Klebsiella. Genomic analysis is under way to identify all members in END-49 involved in the biotransformation and the actual pathway leading to END-production.
Biotransformation is a very economic, efficient and environmentally friendly way of mass-producing enterodiol from defatted flaxseeds.
Bacteriocins are antimicrobial proteins and peptides ribosomally synthesized by some bacteria which can effect both intraspecies and interspecies killing.
Moraxella catarrhalis strain E22 containing plasmid pLQ510 was shown to inhibit the growth of M. catarrhalis strain O35E. Two genes (mcbA and mcbB) in pLQ510 encoded proteins predicted to be involved in the secretion of a bacteriocin. Immediately downstream from these two genes, a very short ORF (mcbC) encoded a protein which had some homology to double-glycine bacteriocins produced by other bacteria. A second very short ORF (mcbI) immediately downstream from mcbC encoded a protein which had no significant similarity to other proteins in the databases. Cloning and expression of the mcbI gene in M. catarrhalis O35E indicated that this gene encoded the cognate immunity factor. Reverse transcriptase-PCR was used to show that the mcbA, mcbB, mcbC, and mcbI ORFs were transcriptionally linked. This four-gene cluster was subsequently shown to be present in the chromosome of several M. catarrhalis strains including O12E. Inactivation of the mcbA, mcbB, or mcbC ORFs in M. catarrhalis O12E eliminated the ability of this strain to inhibit the growth of M. catarrhalis O35E. In co-culture experiments involving a M. catarrhalis strain containing the mcbABCI locus and one which lacked this locus, the former strain became the predominant member of the culture after overnight growth in broth.
This is the first description of a bacteriocin and its cognate immunity factor produced by M. catarrhalis. The killing activity of the McbC protein raises the possibility that it might serve to lyse other M. catarrhalis strains that lack the mcbABCI locus, thereby making their DNA available for lateral gene transfer.
Iron is recognized as an important trace element, essential for most organisms including pathogenic bacteria. HugZ, a protein related to heme iron utilization, is involved in bacterial acquisition of iron from the host. We previously observed that a hugZ homologue is correlated with the adaptive colonization of Helicobacter pylori (H. pylori), a major gastro-enteric pathogen. However, its exact physiological role remains unclear.
A gene homologous to hugZ, designated hp0318, identified in H. pylori ATCC 26695, exhibits 66% similarity to cj1613c of Campylobacter jejuni NCTC 11168. Soluble 6 × His fused-HugZ protein was expressed in vitro. Hemin-agrose affinity analysis indicated that the recombinant HugZ protein can bind to hemin. Absorption spectroscopy at 411 nm further revealed a heme:HugZ binding ratio of 1:1. Enzymatic assays showed that purified recombinant HugZ protein can degrade hemin into biliverdin and carbon monoxide in the presence of either ascorbic acid or NADPH and cytochrome P450 reductase. The biochemical and enzymatic characteristics agreed closely with those of Campylobacter jejuni Cj1613c protein, implying that hp0318 is a functional member of the HugZ family. A hugZ deletion mutant was obtained by homologous recombination. This mutant strain showed poor growth when hemoglobin was provided as the source of iron, partly because of its failure to utilize hemoglobin efficiently. Real-time quantitative PCR also confirmed that the expression of hugZ was regulated by iron levels.
These findings provide biochemical and genetic evidence that hugZ (hp0318) encodes a heme oxygenase involved in iron release/uptake in H. pylori.
Community acquired (CA) methicillin-resistant Staphylococcus aureus (MRSA) increasingly causes disease worldwide. USA300 has emerged as the predominant clone causing superficial and invasive infections in children and adults in the USA. Epidemiological studies suggest that USA300 is more virulent than other CA-MRSA. The genetic determinants that render virulence and dominance to USA300 remain unclear.
We sequenced the genomes of two pediatric USA300 isolates: one CA-MRSA and one CA-methicillin susceptible (MSSA), isolated at Texas Children's Hospital in Houston. DNA sequencing was performed by Sanger dideoxy whole genome shotgun (WGS) and 454 Life Sciences pyrosequencing strategies. The sequence of the USA300 MRSA strain was rigorously annotated. In USA300-MRSA 2658 chromosomal open reading frames were predicted and 3.1 and 27 kilobase (kb) plasmids were identified. USA300-MSSA contained a 20 kb plasmid with some homology to the 27 kb plasmid found in USA300-MRSA. Two regions found in US300-MRSA were absent in USA300-MSSA. One of these carried the arginine deiminase operon that appears to have been acquired from S. epidermidis. The USA300 sequence was aligned with other sequenced S. aureus genomes and regions unique to USA300 MRSA were identified.
USA300-MRSA is highly similar to other MRSA strains based on whole genome alignments and gene content, indicating that the differences in pathogenesis are due to subtle changes rather than to large-scale acquisition of virulence factor genes. The USA300 Houston isolate differs from another sequenced USA300 strain isolate, derived from a patient in San Francisco, in plasmid content and a number of sequence polymorphisms. Such differences will provide new insights into the evolution of pathogens.
Although more than 100 Chlamydia pneumoniae hypothetical proteins have been predicted to be inclusion membrane proteins, only a few have been experimentally demonstrated to be in the inclusion membrane. Using antibodies raised with fusion proteins, we characterized four such hypothetical proteins encoded by two gene clusters (Cpn0146-147 and Cpn0284-285) in the C. pneumoniae genome.
Cpn0146 and 0147 were detected in the inclusion membrane while Cpn0284 and 0285 inside inclusion and mainly associated with reticulate bodies although all four proteins contain an N-terminal bi-lobed hydrophobic region, a signature motif assigned to inclusion membrane proteins. These four hypothetical proteins were only detected in cells infected with C. pneumoniae but not other chlamydial species, with Cpn0147 at 6 hours and Cpn0146, 0284 & 0285 at 24 hours after infection. Cpn0146 & 147 but not Cpn0284 and 285 co-localized with a host cell endoplasmic reticulum marker, a property known to be possessed by some chlamydial inclusion membrane proteins, when expressed in the host cell cytosol via transgenes. However, the endoplasmic reticulum localization of the C. pneumoniae inclusion membrane proteins did not result in inhibition of the subsequent C. pneumoniae infection.
The hypothetical proteins Cpn0146 & 0147 were localized in the C. pneumoniae inclusion membrane while Cpn0284 & 0285 within the inclusion although all four were predicted to be Inc proteins, suggesting the need to experimentally characterize the predicted Inc proteins.
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.
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Magnaporthe oryzae; Conidiogenesis; Stress response; Cell wall integrity; Pathogenesis
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.
Yersinia; CsrA; Csr system; Motility; Salt sensitivity; Antibiotic sensitivity; Temperature sensitivity; Psychrotroph; Mutant selection
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
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crdR; Curdlan; Agrobacterium; Transcriptional regulator
Human immunodeficiency virus type 1 (HIV-1) Vif hijacks an E3 ligase to suppress natural APOBEC3 restriction factors, and core binding factor β (CBF-β) is required for this process. Although an extensive region of Vif spanning most of its N-terminus is known to be critical for binding with CBF-β, involvement of the Vif C-terminus in the interaction with CBF-β has not been fully investigated.
Here, through immunoprecipitation analysis of Vif C-terminal truncated mutants of various lengths, we identified that CBF-β binding requires not only certain amino acids (G126A, E134A, Y135A and G138A) in the HCCH region but also the HCCH motif itself, which also affects the Vif-mediated suppression of APOBEC3G/APOBEC3F (A3G/A3F). These mutants still maintained interactions with substrate A3G or A3F as well as other cellular factors ElonginB/C (ELOB/C), indicating that their structures were not functionally affected. Moreover, by determining that the BC box also is necessary for CBF-β interaction in vivo, we speculate that binding to ELOB/C induces conformational changes in Vif, facilitating its interaction with CBF-β and consequent interaction with CUL5.
These results provide important information on the assembly of the Vif-CUL5-E3 ubiquitin ligase. Identification of the new binding interface with CBF-β at the C-terminus of HIV-1 Vif also provides novel targets for the development of HIV-1 inhibitors.
HIV-1 Vif; CBF-β; C-terminus; APOBEC3
Porcine reproductive and respiratory syndrome virus (PRRSV) is a continuous threat to the pig industry, causing high economic losses worldwide. Current vaccines have specific limitations in terms of their safety and efficacy, so the development of novel antiviral drugs is urgently required. The aim of this study was to evaluate the inhibitory effects and underlying molecular mechanisms of the antimicrobial peptide cecropin P1 (CP1) against PRRSV infection in vitro.
CP1 not only displayed extracellular virucidal activity against PRRSV, but also exerted a potent inhibitory effect when added either before, simultaneously with, or after viral inoculation. The inhibitory effect of CP1 occurred during viral attachment, but not at viral entry into Marc-145 cells. CP1 also inhibited viral particle release and attenuated virus-induced apoptosis during the late phase of infection. CP1 exerted similar inhibitory effects against PRRSV infection in porcine alveolar macrophages, the cells targeted by the virus in vivo during its infection of pigs. The expression of interleukin 6 was elevated by CP1 in porcine alveolar macrophages, which might contribute to its inhibition of PRRSV infection.
Collectively, our findings provide a new direction for the development of potential therapeutic drugs against PRRSV infection.
Cecropin P1; PRRSV; Antiviral activity; Antimicrobial peptide
The emerged human infection with avian influenza A (H7N9) virus in China since 2013 has aroused global concerns. There is great demand for simple and rapid diagnostic method for early detection of H7N9 to provide timely treatment and disease control. The aim of the current study was to develop a rapid, accurate and feasible reverse-transcription loop-mediated isothermal amplification (RT-LAMP) assay for detection of H7N9 virus.
The detection limits of the H7- and N9-specific RT-LAMP assay were both approximately 0.2 PFU per reaction. No cross-reactivity was observed with other subtype of influenza viruses or common respiratory viral pathogens. The assay worked well with clinical specimens from patients and chickens, and exhibited high specificity and sensitivity.
The H7/N9 specific RT-LAMP assay was sensitive and accurate, which could be a useful alternative in clinical diagnostics of influenza A (H7N9) virus, especially in the hospitals and laboratories without sophisticated diagnostic systems.
Influenza virus; H7N9 subtype; Reverse transcription-loop-mediated isothermal amplification; Molecular diagnosis
Acidithiobacillus thiooxidans (A. thiooxidans), a chemolithoautotrophic extremophile, is widely used in the industrial recovery of copper (bioleaching or biomining). The organism grows and survives by autotrophically utilizing energy derived from the oxidation of elemental sulfur and reduced inorganic sulfur compounds (RISCs). However, the lack of genetic manipulation systems has restricted our exploration of its physiology. With the development of high-throughput sequencing technology, the whole genome sequence analysis of A. thiooxidans has allowed preliminary models to be built for genes/enzymes involved in key energy pathways like sulfur oxidation.
The genome of A. thiooxidans A01 was sequenced and annotated. It contains key sulfur oxidation enzymes involved in the oxidation of elemental sulfur and RISCs, such as sulfur dioxygenase (SDO), sulfide quinone reductase (SQR), thiosulfate:quinone oxidoreductase (TQO), tetrathionate hydrolase (TetH), sulfur oxidizing protein (Sox) system and their associated electron transport components. Also, the sulfur oxygenase reductase (SOR) gene was detected in the draft genome sequence of A. thiooxidans A01, and multiple sequence alignment was performed to explore the function of groups of related protein sequences. In addition, another putative pathway was found in the cytoplasm of A. thiooxidans, which catalyzes sulfite to sulfate as the final product by phosphoadenosine phosphosulfate (PAPS) reductase and adenylylsulfate (APS) kinase. This differs from its closest relative Acidithiobacillus caldus, which is performed by sulfate adenylyltransferase (SAT). Furthermore, real-time quantitative PCR analysis showed that most of sulfur oxidation genes were more strongly expressed in the S0 medium than that in the Na2S2O3 medium at the mid-log phase.
Sulfur oxidation model of A. thiooxidans A01 has been constructed based on previous studies from other sulfur oxidizing strains and its genome sequence analyses, providing insights into our understanding of its physiology and further analysis of potential functions of key sulfur oxidation genes.
Acidithiobacillus thiooxidans; Whole genome sequence; Bioinformatics analysis; Real-time quantitative PCR; Sulfur oxidation model
Economically, Leuconostoc lactis is one of the most important species in the genus Leuconostoc. It plays an important role in the food industry including the production of dextrans and bacteriocins. Currently, traditional molecular typing approaches for characterisation of this species at the isolate level are either unavailable or are not sufficiently reliable for practical use. Multilocus sequence typing (MLST) is a robust and reliable method for characterising bacterial and fungal species at the molecular level. In this study, a novel MLST protocol was developed for 50 L. lactis isolates from Mongolia and China.
Sequences from eight targeted genes (groEL, carB, recA, pheS, murC, pyrG, rpoB and uvrC) were obtained. Sequence analysis indicated 20 different sequence types (STs), with 13 of them being represented by a single isolate. Phylogenetic analysis based on the sequences of eight MLST loci indicated that the isolates belonged to two major groups, A (34 isolates) and B (16 isolates). Linkage disequilibrium analyses indicated that recombination occurred at a low frequency in L. lactis, indicating a clonal population structure. Split-decomposition analysis indicated that intraspecies recombination played a role in generating genotypic diversity amongst isolates.
Our results indicated that MLST is a valuable tool for typing L. lactis isolates that can be used for further monitoring of evolutionary changes and population genetics.
Aflatoxins (AFs) are potent carcinogenic compounds produced by several Aspergillus species, which pose serious threats to human health. As sugar is a preferred carbohydrate source for AF production, we examined the possibility of using sugar analogs to inhibit AF biosynthesis.
We showed that although D-glucal cannot be utilized by A. flavus as the sole carbohydrate source, it inhibited AF biosynthesis and promoted kojic acid production without affecting mycelial growth when applied to a glucose-containing medium. The inhibition occurred before the production of the first stable intermediate, norsolorinic acid, suggesting a complete inhibition of the AF biosynthetic pathway. Further studies showed that exogenous D-glucal in culture led to reduced accumulation of tricarboxylic acid (TCA) cycle intermediates and reduced glucose consumption, indicating that glycolysis is inhibited. Expression analyses revealed that D-glucal suppressed the expression of AF biosynthetic genes but promoted the expression of kojic acid biosynthetic genes.
D-glucal as a non-metabolizable glucose analog inhibits the AF biosynthesis pathway by suppressing the expression of AF biosynthetic genes. The inhibition may occur either directly through interfering with glycolysis, or indirectly through reduced oxidative stresses from kojic acid biosynthesis.
D-glucal; D-galactal; Aflatoxin biosynthesis; Aspergillus flavus; Metabolomics
Candida albicans can form biofilms on intravenous catheters; this process plays a key role in the pathogenesis of catheter infections. This study evaluated the effect of human serum (HS) on C. albicans biofilm formation and the expression of adhesion-related genes in vitro. A C. albicans laboratory strain (ATCC90028) and three clinical strains were grown for 24 h in RPMI 1640 supplemented with HS or RPMI 1640 alone (as a control). The growth of biofilm cells of four strains was monitored by a Live Cell Movie Analyzer, and by XTT reduction assay. The expression of the adhesion-related genes BCR1, ALS1, ALS3, HWP1 and ECE1 was analyzed by RT-PCR at three time points (60 min, 90 min, and 24 h).
In the adhesion phase, C. albicans cells kept a Brownian movement in RPMI medium containing HS until a large number of germ tubes were formed. In the control group, C. albicans cells quickly adhered to the bottom of the reaction plate. Compared with RPMI 1640, medium supplemented with 3–50% HS caused a significant decrease in biofilm development (all p < 0.001). However, the presence of HS had no significant inhibitory effect on the pre-adhered biofilms (all p > 0.05). Biofilm formation was also inhibited by heat-inactivated and proteinase K pre-treated HS. The presence of 50% HS did not significantly affect the planktonic growth of C. albicans (p > 0.05). At three time points, HS inhibited expression of the ALS1 and ALS3 genes and promoted expression of the HWP1 and ECE1 genes. Significant up-regulation of BCR1 was observed only at the 90-min point.
Human serum reduces biofilm formation by inhibiting the adhesion of C. albicans cells. This response may be associated with the down-regulation of adhesion-related genes ALS1, ALS3 and BCR1. The inhibitory serum component is protease-resistant and heat stable.
Human serum; Biofilm; Adhesion; Candida albicans
Successful viral infection requires the involvement of host cellular factors in their life cycle. Heat shock protein 70 (HSP70) can be recruited by numerous viruses to promote the folding, maturation, or assembly of viral proteins. We have previously shown that HSP70 is significantly elevated in porcine reproductive and respiratory syndrome virus (PRRSV)-infected lungs, suggesting HSP70 may play a potential role during PRRSV infection. In this study, we tried to investigate the role of HSP70 during PRRSV infection.
In this study, we observed that PRRSV infection induced the expression of HSP70. The down-regulation of HSP70 using quercetin, a HSPs synthesis inhibitor, or small interfering RNAs (siRNA) reduced the viral protein level and viral production. Notably, these inhibitory effects on PRRSV infection could be attenuated by heat shock treatment. In addition, HSP70 was found to colocalize with the viral double-stranded RNA (dsRNA) and knockdown of HSP70 decreased the dsRNA levels, suggesting HSP70 is involved in the formation of viral replication and transcription complex (RTC) and thus affects the viral replication.
Our study revealed that HSP70 is an essential host factor required for the replication of PRRSV. The inhibition of HSP70 significantly reduced PRRSV replication, which may be applied as an effective antiviral strategy.
PRRSV; HSP70; DsRNA; Replication; Antiviral
Rhodosporidium toruloides is a β-carotenoid accumulating, oleaginous yeast that has great biotechnological potential. The lack of reliable and efficient genetic manipulation tools have been a major hurdle blocking its adoption as a biotechnology platform.
We report for the first time the development of a highly efficient targeted gene deletion method in R. toruloides ATCC 10657 via Agrobacterium tumefaciens-mediated transformation. To further improve targeting frequency, the KU70 and KU80 homologs in R. toruloides were isolated and characterized in detail. A KU70-deficient mutant (∆ku70e) generated with the hygromycin selection cassette removed by the Cre-loxP recombination system showed a dramatically improved targeted gene deletion frequency, with over 90% of the transformants being true knockouts when homology sequence length of at least 1 kb was used. Successful gene targeting could be made with homologous flanking sequences as short as 100 bp in the ∆ku70e strain. KU70 deficiency did not perturb cell growth although an elevated sensitivity to DNA mutagenic agents was observed. Compared to the other well-known oleaginous yeast, Yarrowia lipolytica, R. toruloides KU70/KU80 genes contain much higher density of introns and are the most GC-rich KU70/KU80 genes reported.
The KU70-deficient mutant generated herein was effective in improving gene deletion frequency and allowed shorter homology sequences to be used for gene targeting. It retained the key oleaginous and fast growing features of R. toruloides. The strain should facilitate both fundamental and applied studies in this important yeast, with the approaches taken here likely to be applicable in other species in subphylum Pucciniomycotina.
Nonhomologous end-joining (NHEJ); Rhodosporidium toruloides; Oleaginous yeast; Agrobacterium tumefaciens-mediated transformation (ATMT); Homologous recombination