an-approach-to-constructing-multispecies-biofilm-communities-from

An Approach to Constructing Multispecies Biofilm Communities from Rhizosphere Soil

The multispecies biofilm is a naturally occurring and dominant lifestyle of bacteria in nature, including in rhizosphere soil, although the current ...

Huazhong Agricultural University

Transcriptional regulation plays a critical role during the infection of Mycobacterium tuberculosis, the causative agent of tuberculosis. A two-component system, PhoPR, is clearly involved in the regulation of pathogenic virulence and persistence. However, the regulatory mechanism, as well as the regulator, of the phoPR operon remains uncharacterized in M. tuberculosis and its related species thus far. In the present study, we characterize an ArsR transcriptional factor, corresponding to Rv2034 and Ms6762 in M. tuberculosis and Mycobacterium smegmatis, respectively, as the first regulator of phoP in both mycobacterial species. The interaction between ArsR regulator and target promoters is conserved in these two mycobacterial species, and an inverted repeat sequence motif is successfully mapped out for the recognition of ArsR. Utilizing lacZ reporter genes and overexpression analysis, the ArsR regulator is shown to positively regulate the expression of phoP in M. smegmatis, different from most ArsR family regulators generally as a repressor. The current study establishes a direct link between the ArsR transcriptional factor and the regulation of phoP in mycobacteria. Our findings imply that ArsR may be involved in the pathogenesis of M. tuberculosis through its regulation of the phoPR operon.

An ArsR-like transcriptional factor recognizes a conserved sequence motif and positively regulates the expression of phoP in mycobacteria.

The genome of Mycobacterium tuberculosis, the causative agent of tuberculosis, encodes a large number of putative transcriptional regulators. However, the identity and target genes of only a few of them have been clearly identified to date. In a recent study, the ArsR family regulator Rv2034 was characterized as a novel positive regulator of phoP. In the current study, we characterized the auto-repressive capabilities of Rv2034 and identified several residues in the protein critical for its DNA binding activities. We also provide evidence that Rv2034 forms dimers in vitro. Furthermore, by using DNaseI footprinting assays, a palindromic sequence was identified as its binding site. Notably, we found that the dosR promoter region contains the binding motif for Rv2034, and that Rv2034 positively regulates the expression of the dosR gene. The potential roles of Rv2034 in the regulation of lipid metabolism and hypoxic adaptation are discussed.

Characterization of a novel ArsR-like regulator encoded by Rv2034 in Mycobacterium tuberculosis.

Toxin-antitoxin (TA) systems play significant roles in the regulation of bacterial growth and persistence, and their functions usually depend on protein-protein interaction between their constituent TA proteins. However, the regulatory mechanisms of these systems, particularly their interaction with other cellular components, are still poorly understood. This study investigated cross-talk between the TA module RelJ/K (Rv3357/Rv3358) and the transcriptional regulator staphylococcal iron regulator repressor (SirR, Rv2788) from Mycobacterium tuberculosis. We characterized the physical interaction of SirR with both RelJ and RelK using bacterial two-hybrid, pull-down and co-immunoprecipitation assays. Similarly to RelK, SirR regulates the DNA-binding activity of RelJ and alleviates its inhibitory effect on the activity of the Rv3357p promoter. Furthermore, SirR may replace RelJ to alleviate the inhibitory effect of the toxin RelK on bacterial growth. Conversely, both RelJ and RelK competitively inhibit the interaction between SirR and their respective promoters. Thus, our results show that SirR interacts with a pair of toxin and antitoxin proteins, and exhibits antitoxin-like function to neutralize the toxin. These findings demonstrate a novel function of the SirR regulator of M. tuberculosis as well as a novel mechanism of regulation of TA systems.

Characterization of the interaction between a SirR family transcriptional factor of Mycobacterium tuberculosis, encoded by Rv2788, and a pair of toxin-antitoxin proteins RelJ/K, encoded by Rv3357 and Rv3358.

Isoniazid (INH), an anti-tuberculosis (TB) drug, has been widely used for nearly 60 years. However, the pathway through which Mycobacterium tuberculosis responds INH remain largely unclear. In this study, we characterized a novel transcriptional factor, InbR, which is encoded by Rv0275c and belongs to the TetR family, that is directly responsive to INH. Disrupting inbR made mycobacteria more sensitive to INH, whereas overexpressing inbR decreased bacterial susceptibility to the drug. InbR could bind specifically to the upstream region of its own operon at two inverted repeats and act as an auto-repressor. Furthermore, InbR directly bind with INH, and the binding reduced InbR's DNA-binding ability. Interestingly, susceptibilities were also changed by InbR for other anti-TB drugs, such as rifampin, implying that InbR may play a role in multi-drug resistance. Additionally, microarray analyses revealed a portion genes of the inbR regulon have similar expression patterns in inbR-overexpressing strain and INH-treated wild type strain, suggesting that these genes, for example iniBAC, may be responsible to the drug resistance of inbR-overexpressing strain. The regulation of these genes by InbR were further assessed by ChIP-seq assay. InbR may regulate multiple drug resistance of mycobacteria through the regulation of these genes.

InbR, a TetR family regulator, binds with isoniazid and influences multidrug resistance in Mycobacterium bovis BCG.

Clay minerals and metal oxides, as important parts of the soil matrix, play crucial roles in the development of microbial communities. However, the mechanism underlying such a process, particularly on the formation of soil biofilm, remains poorly understood. Here, we investigated the effects of montmorillonite, kaolinite, and goethite on the biofilm formation of the representative soil bacteria . The bacterial biofilm formation in goethite was found to be impaired in the initial 24 h but burst at 48 h in the liquid-air interface. Confocal laser scanning microscopy showed that the biofilm biomass in goethite was 3-16 times that of the control, montmorillonite, and kaolinite at 48 h. Live/Dead staining showed that cells had the highest death rate of 60% after 4 h of contact with goethite, followed by kaolinite and montmorillonite. Atomic force microscopy showed that the interaction between goethite and bacteria may injure bacterial cells by puncturing cell wall, leading to the swarming of bacteria toward the liquid-air interface. Additionally, the expressions of  and , key players in regulating the biofilm formation, were upregulated at 24 h and downregulated at 48 h in goethite, indicating the initial adaptation of the cells to minerals. A model was proposed to describe the effects of goethite on the biofilm formation. Our findings may facilitate a better understanding of the roles of soil clays in biofilm development and the manipulation of bacterial compositions through controlling the biofilm in soils.

 biofilm development in the presence of soil clay minerals and iron oxides.

Bacterial social interaction is a potential influencing factor in determining the fate of invading pathogens in diverse environments. In this study, interactions between two representative resident species (Bacillus subtilis and Pseudomonas putida) and a leading food-borne disease causative pathogen (Vibrio parahaemolyticus) were examined. An antagonistic effect toward V. parahaemolyticus was observed for B. subtilis but not for P. putida. However, the relative richness of the pathogen remained rather high in B. subtilis co-cultures and was, unexpectedly, not sensitive to the initial inoculation ratios. Furthermore, two approaches were found to be efficient at modulating the relative richness of the pathogen. (1) The addition of trace glycerol and manganese to Luria-Bertani medium (LBGM) reduced the richness of V. parahaemolyticus in the co-culture with B. subtilis and in contrast, increased its richness in the co-culture with P. putida, although it did not affect the growth of V. parahaemolyticus by its own. (2) The relative richness of V. parahaemolyticus on semisolid medium decreased significantly as a function of an agar gradient, ranging from 0 to 2%. Furthermore, we explored the molecular basis of bacterial interaction through transcriptomic analysis. In summary, we investigated the interactions between a pathogen invader and two resident bacteria species, showing that the different influences on a pathogen by different types of interactions can be modulated by chemicals and medium fluidity.

Divergent Influence to a Pathogen Invader by Resident Bacteria with Different Social Interactions.

The ferric uptake regulator A (FurA) plays an essential role in responding to oxidative stress in mycobacteria. The genome of Mycobacterium smegmatis harbours three FurA orthologs; however, the potential cross-talk and contribution to drug resistance of different furA operon remain underdetermined. In this study, we characterized the cross-regulation and effect in drug resistance of these orthologs from M. smegmatis. Cross-binding of FurA protein to furA promoter was observed. The binding of FurA1 to furA3p and FurA2 to furA1p or furA3p is even more pronounced than their self-binding. The three FurA proteins are all functional at repressing the expression of the peroxidase enzyme katG1/katG2 in vivo. When overexpressing any of the furA orthologs in M. smegmatis, the bacteria become more resistant to isoniazid (INH). This pattern is consistent with that in Mycobacterium bovis. However, the knockdown of furA does not affect the INH sensitivity. This is the first report of cross-talk and contribution to drug resistance of all three furA orthologs in M. smegmatis.

Cross-talk between the three furA orthologs in Mycobacterium smegmatis and the contribution to isoniazid resistance.

The bacteria drug resistance is not only associated with the gain of drug resistance gene but also relied on the adaptation of bacterial cells to antibiotics by transcriptional regulation. However, only a few transcription factors that regulate drug resistance have been characterized in mycobacteria. In this study, a TetR family transcriptional factor (OxiR), encoded by Rv0067c in Mycobacterium tuberculosis, was found to be an isoniazid (INH) resistance regulator. Comparing with the wild-type strain, the oxiR overexpressing strain is four times resistant to INH, whereas the oxiR knockout strain is eight times sensitive to INH. However, the rifamycin and ethambutol resistance were not influenced by oxiR. OxiR can bind to self-promoter at a 66 bp imperfect palindromic motifs. Interestingly, OxiR directly binds to INH, and thereby alleviate the self-repression. Furthermore, OxiR negatively regulated an oxidoreductase encoded by Rv0068. And the susceptibility of the Rv0068-overexpressing and oxiR knockout strains to all the three above-mentioned anti-tuberculosis drugs was equivalent, suggesting that the effect of oxiR to INH susceptibility is attributed to the derepression of Rv0068. In conclusion, we showed that OxiR can specifically modulate INH susceptibility by regulating an oxidoreductase encoding gene, both of which have not been associated with drug-resistance previously.

OxiR specifically responds to isoniazid and regulates isoniazid susceptibility in mycobacteria.

Polymerase chain reaction (PCR) is used as an in vitro model system of DNA replication to assess the genotoxicity of nanoparticles (NPs). Prior results showed that several types of NPs inhibited PCR efficiency and increased amplicon error frequency. In this study, we examined the effects of various metal oxide NPs on inhibiting PCR, using high- vs. low-fidelity DNA polymerases; we also examined NP-induced DNA mutation bias at the single nucleotide level. The effects of seven major types of metal oxide NPs (FeO, ZnO, CeO, FeO, AlO, CuO, and TiO) on PCR replication via a low-fidelity DNA polymerase (Ex Taq) and a high-fidelity DNA polymerase (Phusion) were tested. The successfully amplified PCR products were subsequently sequenced using high-throughput amplicon sequencing. Using consistent proportions of NPs and DNA, we found that the effects of NPs on PCR yield differed depending on the DNA polymerase. Specifically, the efficiency of the high-fidelity DNA polymerase (Phusion) was significantly inhibited by NPs during PCR; such inhibition was not evident in reactions with Ex Taq. Amplicon sequencing showed that the overall error rate of NP-amended PCR was not significantly different from that of PCR without NPs ( > 0.05), and NPs did not introduce single nucleotide polymorphisms during PCR. Thus, overall, NPs inhibited PCR amplification in a DNA polymerase-specific manner, but mutations were not introduced in the process.

Impact of metal oxide nanoparticles on in vitro DNA amplification.

It is urgent to understand the regulatory mechanism of drug resistance in widespread bacterial pathogens. In Mycobacterium tuberculosis, several transcriptional regulators have been found to play essential roles in regulating its drug resistance. In this study, we found that an ArsR family transcription regulator encoded by Rv2642 (CdiR) responds to isoniazid (INH), a widely used anti-tuberculosis (TB) drug. CdiR negatively regulates self and adjacent genes, including arsC (arsenic-transport integral membrane protein ArsC). CdiR directly interacts with INH and Cd(II). The binding of INH and Cd(II) both reduce its DNA-binding activity. Disrupting cdiR increased the drug susceptibility to INH, whereas overexpressing cdiR decreased the susceptibility. Strikingly, overexpressing arsC increased the drug susceptibility as well as cdiR. Additionally, both changes in cdiR and arsC expression caused sensitivity to other drugs such as rifamycin and ethambutol, where the minimal inhibitory concentrations in the cdiR deletion strain were equal to those of the arsC-overexpressing strain, suggesting that the function of CdiR in regulating drug resistance primarily depends on arsC. Furthermore, we found that Cd(II) enhances bacterial resistance to INH in a CdiR-dependent manner. As a conclusion, CdiR has a critical role in directing the interplay between Cd(II) metal ions and drug susceptibility in mycobacteria.

Cd(II)-binding transcriptional regulator interacts with isoniazid and regulates drug susceptibility in mycobacteria.

Coculture is an important model system in microbial ecology studies. As a key experimental parameter, the initial inoculation ratio has a crucial impact on the results of the coculture system. However, such an effect has never been investigated under multiple niche conditions. In this study, we established a simple coculture system with two model bacteria in various carbon sources and investigated the influence of initial inoculum ratios of 1:1000 to 1000:1 on community structure, function, and bacterial interaction. We found that the final ratio of the cocultures with different initial inoculum ratios differed in approximately five-sixths of the carbon sources, suggesting that the final ratio is highly dependent on the initial inoculum ratio, while the carbon source preferences of bacteria could not predict the final ratio of cocultures. Furthermore, we found that the initial ratio could regulate the metabolic capacity of the coculture, as only cocultures with initial ratios of 1:1 and 1000:1 gained high capacity on 14 specific carbon sources. The underlying reason may be that the pattern of species interaction is changed by the initial ratio. In conclusion, we showed that the initial ratio can induce emergent properties in coculture. These findings suggest that the initial ratio not only impacts the reproducibility of coculture experiments but also can influence our understanding of generic microbial ecology.

The initial inoculation ratio regulates bacterial coculture interactions and metabolic capacity.

Venn diagrams are widely used diagrams to show the set relationships in biomedical studies. In this study, we developed ggVennDiagram, an R package that could automatically generate high-quality Venn diagrams with two to seven sets. The ggVennDiagram is built based on ggplot2, and it integrates the advantages of existing packages, such as venn, RVenn, VennDiagram, and sf. Satisfactory results can be obtained with minimal configurations. Furthermore, we designed comprehensive objects to store the entire data of the Venn diagram, which allowed free access to both intersection values and Venn plot sub-elements, such as set label/edge and region label/filling. Therefore, high customization of every Venn plot sub-element can be fulfilled without increasing the cost of learning when the user is familiar with ggplot2 methods. To date, ggVennDiagram has been cited in more than 10 publications, and its source code repository has been starred by more than 140 GitHub users, suggesting a great potential in applications. The package is an open-source software released under the GPL-3 license, and it is freely available through CRAN (https://cran.r-project.org/package=ggVennDiagram).

ggVennDiagram: An Intuitive, Easy-to-Use, and Highly Customizable R Package to Generate Venn Diagram.

The CRISPR-Cas system is an adaptive immune system for many bacteria and archaea to defend against foreign nucleic acid invasion, and this system is conserved in the genome of  (). Although the CRISPR-Cas system-mediated immune defense mechanism has been revealed in , the regulation of  gene expression is poorly understood. In this study, we identified a transcription factor, CasR (CRISPR-associated protein repressor, encoded by ), and it could bind to the upstream DNA sequence of the CRISPR-Cas gene cluster and regulate the expression of  genes. EMSA and ChIP assays confirmed that CasR could interact with the upstream sequence of the  promoter, both in vivo and in vitro. Furthermore, DNA footprinting assay revealed that CasR recognized a 20 bp palindromic sequence motif and negatively regulated the expression of  In conclusion, our research elucidates the regulatory effect of CasR on the expression of CRISPR-associated genes in mycobacteria, thus providing insight into gene expression regulation of the CRISPR-Cas system.

Regulation of CRISPR-Associated Genes by Rv1776c (CasR) in .

Soil microbial diversity is important for maintaining ecosystem functions. However, the linkage between microbial diversity, especially rare and abundant bacterial diversity, and carbon decomposition remains largely unknown. In this study, we assessed the establishment and maintenance of rare and abundant bacterial α-diversities at the taxonomic and phylogenetic levels and their linkages with soil carbon decomposition separately in four Chinese woodlands. Compared to abundant bacteria, rare bacteria showed higher community diversity, tighter phylogenetic clustering, wider environmental breadth, stronger phylogenetic signals, and higher functional redundancy. The assembly of the abundant bacterial subcommunity was governed by stochastic (59.2%) and deterministic (41.8%) processes, whereas the assembly of the rare bacterial subcommunity was mainly dominated by deterministic processes (85.8%). Furthermore, total phosphorus, soil pH, and ammonium nitrogen balanced stochastic and deterministic processes in both rare and abundant bacterial subcommunities. Our results reveal that rare bacteria displayed stronger environmental adaptability and environmental constraint. Importantly, the α-diversities of rare taxa, rather than abundant taxa, were significantly related to carbon decomposition. This study provides a holistic understanding of biogeographic patterns of abundant and rare bacteria and their α-diversities in relation to carbon decomposition, thus helping us better predict and regulate carbon dynamics under the background of global climate change.

Stronger linkage of diversity-carbon decomposition for rare rather than abundant bacteria in woodland soils.

Taxonomic convergence is common in bacterial communities but its underlying molecular mechanism remains largely unknown. We thus conducted a time-series transcriptional analysis of a convergent two-species synthetic community that grew in a closed broth-culture system. By analyzing the gene expression and monitoring the community structure, we found that gene expression mainly changed in the early stage, whereas community structure significantly changed in the late stage. The significant change of gene expression occurred even at the very beginning, which was designated as "0 h effect", suggesting the effect of species interaction on gene expression was inevitable. Besides, the effect of interaction on gene expression has a "population effect", which means that majority species have greater impact on gene expressions of minority species than vice versa. Furthermore, gene set enrichment analysis revealed that among a total of 63 unique pathways (occupying about 50% of all the metabolic pathways in both species), 40 (63%) were consistently suppressed, 16 (25%) were conditionally expressed, and only 7 (11%) were consistently activated. Overall, they were strictly regulated by both time and initial structures. Therefore, we proposed that microorganism responses and the induced gene expression changes play important roles in the process of community succession.

Emergent transcriptional adaption facilitates convergent succession within a synthetic community.

Protection against oxidative stress is a vital defense mechanism for Mycobacterium tuberculosis within the host. However, few transcription factors that control bacterial antioxidant defense are known. Here, we present evidence that SdrR, encoded by the MSMEG_5712 (Ms5712) gene, functions as an oxidative stress response regulator in Mycobacterium smegmatis. SdrR recognizes an 11-bp motif sequence in the operon's upstream regulatory region and negatively regulates the expression of short-chain dehydrogenases/reductases (SDR). Overexpressing sdrR inhibited SDR expression, which rendered the strain oxidative more stress-sensitive. Conversely, sdrR knockout alleviates SDR repression, which increases its oxidative stress tolerance. Thus, SdrR responds to oxidative stress by negatively regulating sdr expression. Therefore, this study elucidated an underlying regulatory mechanism behind mycobacterial oxidative stress adaptation.

Article	Year
An ArsR-like transcriptional factor recognizes a conserved sequence motif and positively regulates the expression of phoP in mycobacteria. Biochemical and biophysical research communications\| PubMed ID: 21782791	2011
Characterization of a novel ArsR-like regulator encoded by Rv2034 in Mycobacterium tuberculosis. PloS one\| PubMed ID: 22558408	2012
Characterization of the interaction between a SirR family transcriptional factor of Mycobacterium tuberculosis, encoded by Rv2788, and a pair of toxin-antitoxin proteins RelJ/K, encoded by R... The FEBS journal\| PubMed ID: 24725376	2014
InbR, a TetR family regulator, binds with isoniazid and influences multidrug resistance in Mycobacterium bovis BCG. Scientific reports\| PubMed ID: 26353937	2015
biofilm development in the presence of soil clay minerals and iron oxides. NPJ biofilms and microbiomes\| PubMed ID: 28649405	2017
Divergent Influence to a Pathogen Invader by Resident Bacteria with Different Social Interactions. Microbial ecology\| PubMed ID: 29858645	2019
Cross-talk between the three furA orthologs in Mycobacterium smegmatis and the contribution to isoniazid resistance. Journal of biochemistry\| PubMed ID: 30993320	2019
OxiR specifically responds to isoniazid and regulates isoniazid susceptibility in mycobacteria. FEMS microbiology letters\| PubMed ID: 31125044	2019
Impact of metal oxide nanoparticles on in vitro DNA amplification. PeerJ\| PubMed ID: 31293839	2019
Cd(II)-binding transcriptional regulator interacts with isoniazid and regulates drug susceptibility in mycobacteria. Journal of biochemistry\| PubMed ID: 32706888	2021
The initial inoculation ratio regulates bacterial coculture interactions and metabolic capacity. The ISME journal\| PubMed ID: 32887945	2021
ggVennDiagram: An Intuitive, Easy-to-Use, and Highly Customizable R Package to Generate Venn Diagram. Frontiers in genetics\| PubMed ID: 34557218	2021
Regulation of CRISPR-Associated Genes by Rv1776c (CasR) in . Biomolecules\| PubMed ID: 36830769	2023
Stronger linkage of diversity-carbon decomposition for rare rather than abundant bacteria in woodland soils. Frontiers in microbiology\| PubMed ID: 36937304	2023
Emergent transcriptional adaption facilitates convergent succession within a synthetic community. ISME communications\| PubMed ID: 37938635	2021
SdrR, a LysR-Type Regulator, responds to the mycobacterial antioxidant defense. Journal of biochemistry\| PubMed ID: 38444151	2024

Chun-hui Gao

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