Accordingly, the absorbance value of crystal violet from wild type was over three times greater than that of the hfq mutant Fig. A cell-counting kit CCK -8 assay was conducted to evaulate the cell viability, and the results demonstrated that cell viability was only slightly inhibited in hfq mutant and no significant difference was found between Xac 29—1 and hfq mutant Data not shown. The tests were repeated three times. For pathogenicity test, wound infection assay in sweet orange leaves was used. Compared with the NB medium, the growth of the hfq mutant almost showed no difference at the low concentration 0.
The resistance of Xac to high and low pH was significantly affected by the mutation of hfq although the growth of complementation strain was also slightly affected at the high and low pH conditions Fig. Taken together, the hfq mutation impairs Xac resistance to H 2 O 2 and pH. Three replicates for each treatment were used, and the experiment was repeated three times. Disruption of hfq significantly changed expression of genes. Of them, genes were down-regulated and 84 genes were up-regulated.
The expression trends of 24 genes were similar with those revealed by RNA-seq results, demonstrating the reliability of RNA-seq analysis. RT-qPCR of 26 selected differentially expressed genes DEGs related with bacterial chemotaxis a , flagellar assembly b , secretion system c and ribosomal protein d.
Vertical bars represent standard errors. Although the inactivation of hfq in different bacterial species has exhibited a pleiotropic phenotype [ 4 , 17 , 18 , 19 ], its deletion in the genus Xanthomonas displayed similar alterations in growth and motility [ 13 , 14 ].
In this study, the mutation in hfq gene resulted in remarkably reduced bacterial growth rate. CheA, CheW and CheR are core proteins of chemosensory pathways which are essential for motility and pathogenicity in many bacteria [ 20 ]. Transcriptome data related to chemotaxis and flagellar assembly strongly supported the biological results of the attenuation of cell motility and biofilm formation Fig. Hfq is a common regulator of virulence in bacteria [ 3 ].
However, the virulence-related defects are not common in plant—pathogenic bacteria and only reported in a few bacteria, i. Hfq significantly regulated type III secretion system and the other related pathogenicity determinants in E. Transcriptome analysis showed that hfq significantly regulated chemotaxis, bacterial secretion systems, two-component system, quorum sensing and flagellar assembly in Xac.
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Regarding the expression profile of the genes associated with secretion systems type I to type VI , those from type IV and type V secretion systems were unaltered, whereas two genes in type I were slighted repressed and 18 genes from type II, III, VI secretion systems were significantly decreased over 2 folds in hfq mutant Additional file 5 : Table S1.
Similar with previous studies on Xoo and Xcv 13, 14 , the virulence of the hfq mutant of Xac also did not affected through wound infection assay. It should be noted that virulence phenotype between Xac wild type and waxcO mutant had no difference by wound infiltration, whereas significant differences in lesion numbers were observed by spray assay [ 22 ]. Non-wound inoculation, spraying or swabbing, remains to be used to check the pathogenicity phenotype of hfq mutant of Xac , Xoo and Xcv.
It is interesting that nine ribosomal protein genes were significantly up-regulated through transcriptome data. Of the nine proteins, RpsE codes for 30S subunit ribosomal protein S5 and the other eight proteins are components of the ribosomal large subunit. It should be note that there was about four-fold expression increase for rplU - rpmA , encodes for 50S subunit r-proteins L21 and L27, respectively Additional file 5 : Table S1.
Although it remains unclear how Hfq affects these ribosomal proteins of Xac , the expression differences of ribosomal proteins, probably resulted in reduced translation accuracy, might also play certain roles in the pleiotropic defects of hfq mutants. Many sRNA candidates in genus Xanthomonas have been generated by high-throughput transcriptome sequencing approaches [ 13 , 14 , 15 ]. It should be noted that the accumulation and activity of only six sRNAs were closely related with hfq whereas some sRNAs involved in virulence, i.
It might partially explain why the hfq mutant strain of Xanthomonas spp. So far, the function of Hfq is still obscure in Xanthomonas spp. In this study, biological analyses of the hfq mutant clearly point toward the requirement for Hfq function in multiple biological processes in Xac , i. RNA-seq data showed that genes were regulated by hfq gene, reflecting its global regulation role in Xac. In particular, the expression of genes associated with bacterial chemotaxis and flagellar assembly were significantly down-regulated in the hfq mutant, consistent with the reduction of swimming and biofilm formation.
The hfq mutant was generated from Xac 29—1 wild type strain by allelic homologous recombination. The plasmid was transformed into wild type strain Xac 29—1 by electroporation. To complement the hfq mutant, DNA fragment containing the entire hfq gene and its upstream promoter was amplified. All the experiments were repeated at least three times.
Biofilms that formed on polystyrene and glass surfaces were examined as previously described [ 28 ]. Excess crystal violet stain was removed to observe a circle of purple material formed on the glass bottle. All the experiments were repeated three times and the average for each strain was checked by t -test. Each group had three wells and all experiments were repeated three times. The resistance assay against H 2 O 2 was performed as described previously [ 29 ] with minor modifications.
H 2 O 2 with a concentration of 0. The full expanded leaves of pineapple sweet orange Citrus sinensis were used as host materials. Five wounds were produced on the back of the leaves with a needle. Each test was repeated at least three times. After cleaning the raw reads, we mapped the clean reads to the complete genome Xac 29—1 strain CP Differentially expression analysis was performed using the DESeq R package 1. The p -values were adjusted for the false discovery rate FDR. A t -test was performed on log 2 -transformed data to identify the genes with significant changes in expression between wild type and mutant strains.
The 16S rRNA gene was used as an endogenous control. Factor fraction required for the synthesis of bacteriophage Qbeta-RNA. Hfq structure, function and ligand binding. Curr Opin Microbiol. Chao Y, Vogel J. The role of Hfq in bacterial pathogens. Characterization of broadly pleiotropic phenotypes caused by an hfq insertion mutation in Escherichia coli K Mol Microbiol. J Bacteriol. Second, the host possesses mechanisms to restrict Mn within the intracellular phagosomal compartment as well as in the extracellular environment.
The mechanisms for both host restriction of Mn and bacterial acquisition of Mn are presented herein. Bacterial pathogens have evolved dedicated import machinery that allows them to compete with host NRAMP1 for Mn within the phagosome. For details regarding the biophysical characteristics of the proteins involved in this process, see the review by Lisher and Giedroc Bacteria encode several broadly conserved classes of importers.
Studies investigating the role of Mn transporters in virulence have been spearheaded by work in obligate or facultative intracellular pathogens. Interestingly, Y. This suggests that Mn importers have varying roles in different infection models. This variation could be due to differences in Mn availability or oxidative stress levels throughout the host. Intracellular pathogens such as Salmonella enterica Typhimurium that reside within the phagosome of macrophages must survive a Mn deplete environment. Host processes to restrict microbial access to Mn within other cellular compartments, including the cytoplasm, have not yet been identified.
Extracellular Mn is sequestered by calprotectin, which is released by neutrophils actively or through cell lysis.
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In contrast, Neisseria meningitidis requires Mn export through MntX for virulence, presumably to prevent Mn toxicity during infection. Host mechanisms to intoxicate bacteria with Mn have not yet been identified. In an experiment designed to reveal in vivo metal conditions experienced by S.
Further experiments revealed that mice deficient in CP have higher bacterial burdens in the liver following intravenous S. Together, these findings define CP as an important host element for limiting Mn availability to S. Calprotectin has a role in defense against multiple pathogens. In contrast, S. The functions of CP are thus important in multiple infection models. Several aspects regarding the contribution of CP to defense against infection remain unknown.
Furthermore, it is not understood whether the processes are interdependent, i. It is likely that additional vertebrate proteins are involved in Mn chelation during infection. Thus, other unidentified factor s are responsible for removing Mn from sites of infection in the kidney. In vitro studies utilizing purified lactoferrin and transferrin will be useful for determining whether these proteins bind Mn with sufficient affinity to restrict microbial access to this metal. Bacterial Mn import systems are important for the virulence of some, but not all tested, extracellular pathogens.
Streptococcus mutans , S. In contrast, inactivation of the Mn importers mntH and zupT has no effect on E. For both avian pathogenic E. Together, these data suggest that Mn import is required for several extracellular pathogens, but redundant metal cation transporters can rescue single mutants and inactivation of multiple transport systems may be required to impair fitness within the host. Mn import systems are thus important for pathogenesis of multiple organisms. However, the cellular requirement for Mn import during infection is not understood. By comparing this selected mutant pool with the pool selected in WT mice, genes important for growth in metal restricted environments could be compared with genes required for other processes.
The necessity for bacterial acquisition systems during infection demonstrates that Mn is required for pathogenesis, but the requirement for Mn during infection is not completely understood. Here we will briefly present recent findings regarding intracellular uses of Mn see Fig. A number of metabolic enzymes in bacteria require Mn, but the implications for pathogenesis have not been explored [please see previous Reviews Jakubovics and Jenkinson, ; Kehres and Maguire, ].
In the presence of hydrogen peroxide, the transcriptional regulator OxyR is oxidized to form an intramolecular disulfide bind that alters the binding affinity of OxyR to the mntH promoter, leading to the activation of mntH transcription. Mn rescues hydrogen peroxide toxicity in E. SOD activity in S. This topic has been thoroughly reviewed Aguirre and Culotta, ; Lisher and Giedroc, Ribonucleotide reductase RNR enzymes are required for the synthesis of deoxyribonucleotides from ribonucleotides and have been used to decipher whether Mn is a preferred enzyme cofactor during infection.
These observations fit well with an earlier report that SsaB, a putative Mn transporter, is required for virulence of S. Together, these findings suggest that the cofactor preference of NrdEF in vivo is Mn. Cellular processes that require Mn have been identified, and the requirement for Mn in oxidative stress resistance and ribonucleotide reductase activity has been thoroughly studied.
However, many questions remain about the subcellular localization and utilization of Mn within bacteria. The intracellular location of Mn has yet to be elucidated and the predominant reservoir of Mn within the cell has not been identified.
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The wide variety of stressors encountered by bacteria has resulted in countless strategies that are used by pathogens to overcome these insults, which we continue to identify. Clearly, a better understanding of these stress response mechanisms may be useful for developing new strategies to combat bacteria that cause certain infectious diseases. This Research Topic aims to highlight our increasing understanding of mechanisms by which bacteria sense and respond to stresses encountered in the host or other environments.
Examples of stress response mechanisms of interest include, but are not limited to those that respond to antimicrobials, host immune responses, or environmental changes. Original research articles, reviews and mini-reviews, perspectives, and manuscripts describing new methods for studying stress response mechanisms are all welcomed. Although this Research Topic was largely planned with human bacterial pathogens in mind, studies on the stress responses of bacteria that make up the microbiome or cause disease in other organisms are also welcome.
Of high interest are studies that describe mechanisms at the molecular level. Important Note : All contributions to this Research Topic must be within the scope of the section and journal to which they are submitted, as defined in their mission statements. Frontiers reserves the right to guide an out-of-scope manuscript to a more suitable section or journal at any stage of peer review.