Vibrio parahaemolyticus is a seafood-borne pathogen that poses a great threat to public health worldwide. It is found in either a planktonic cell or a biofilm form in the natural environment. The
...locus has been the only extensively studied polysaccharide biosynthesis gene cluster involved in biofilm formation for this bacterium. In this study, we found that an additional polysaccharide biosynthesis locus,
, is also necessary for biofilm maturation. The
locus is composed of two operons, and a loss of their expression leads to a defective biofilm phenotype. The transcription of the
locus is under the control of a sigma 54-dependent response regulator, ScvE. In contrast, the quorum-sensing regulator AphA stimulates the expression of the
locus and the
operon found in the
locus. Bioinformatic analyses demonstrated that
loci are divergent and widely distributed among 28 genera, including 26 belonging to the
and 2 within the
. We also determined that all
locus-positive species are water-dwelling. Some strains of
, Aliivibrio salmonicida, Pseudomonas anguilliseptica, Vibrio breoganii, and Vibrio scophthalmi probably acquired
loci through insertion sequences and/or integrase-mediated horizontal gene transfer. Gene duplication and fusion were also detected in some
homologs. Together, our results suggest that the genome of V. parahaemolyticus harbors two distinct polysaccharide biosynthesis loci, which may play a role in fine-tuning biofilm development, and that
loci likely evolved by horizontal gene transfer, gene loss, gene duplication, and fragment fusion.
Polysaccharides are the major component of biofilms, which provide survival advantages for bacteria in aquatic environments. The seafood-borne pathogen V. parahaemolyticus possesses a functionally uncharacterized polysaccharide biosynthesis locus,
. We demonstrated that the
locus is important for biofilm maturation and that
expression is positively regulated by ScvE. Strains from 148 aquatic bacterial species possess
homolog loci. These bacterial species belong to 28 genera, most of which belong to the
class. The evolution and diversification of
loci are likely driven by horizontal gene transfer, gene loss, gene duplication, and fragment fusion. Our results provide new insights into the function and evolution of this widespread polysaccharide biosynthesis locus.
The bacterial second messenger cyclic diguanylate (c-di-GMP) modulates plankton-to-biofilm lifestyle transition of
species through its transcriptional regulatory effector FleQ. FleQ regulates ...transcription of biofilm- and flagellum-related genes in response to c-di-GMP. Through transcriptomic analysis and FleQ-DNA binding assay, this study identified five new target genes of c-di-GMP/FleQ in
, including
,
,
(
),
(
), and
Except
encoding an outer membrane pore protein and
encoding an adenylate cyclase, the functions of the other three genes encoding hypothetical proteins remain unknown. FleQ and c-di-GMP coordinately inhibit transcription of
and
and promote transcription of
,
, and
Both
and
assays show that FleQ binds directly to promoters of the five genes. Further analyses confirm that LapE plays a central role of in the secretion of adhesin LapA and that c-di-GMP/FleQ increases
transcription, thereby promoting adhesin secretion and biofilm formation. The adenylate cyclase CyaA is responsible for synthesis of another second messenger, cyclic AMP (cAMP). FleQ and c-di-GMP coordinate to decrease the content of cAMP, suggesting that c-di-GMP and FleQ coregulate cAMP by modulating
expression. Overall, this study adds five new members to the c-di-GMP/FleQ-regulated gene family and reveals the role of c-di-GMP/FleQ in LapA secretion and cAMP synthesis regulation in
c-di-GMP/FleQ promotes the plankton-to-biofilm lifestyle transition at the transcriptional level via FleQ in
species. Identification of new target genes directly regulated by c-di-GMP/FleQ helps to broaden the knowledge of c-di-GMP/FleQ-mediated transcriptional regulation. Regulation of
by c-di-GMP/FleQ guarantees highly efficient LapA secretion and biofilm formation. The mechanism of negative correlation between c-di-GMP and cAMP in both
and
remains unknown. Our result concerning transcriptional inhibition of
by c-di-GMP/FleQ reveals the mechanism underlying the decrease of cAMP content by c-di-GMP in
.
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•Dissimilatory sulphate reducing bacteria (DSRB) in an upland-paddy field was studied.•Sulfate reducing potential increased in the paddy stage.•Sulfate reducing potential was ...stimulated by straw incorporation.•The α-diversity of DSRB decreased in the paddy stage.•The DSRB structure was associated with pH, phosphorus, sulfate and moisture.
Sulfate reduction is an essential process in the biogeochemical sulfur cycle in soils. It is mainly driven by dissimilatory sulfate-reducing bacteria (DSRB). The responses of the DSRB community in a rapeseed-rice rotation system to the environmental variability caused by the shift from the upland to the paddy stages, and the effects of the long-term straw returning on this shift remains unclear. We surveyed the sulfate reducing potential (SRP) and the structure of the DSRB community by high-throughput sequencing that targeted dsrB (and the xenologous dsrB) in soils. The SRP, ranging from 1.2 to 5.8μmold−1dwg−1, was increased in the paddy stage, and it was likely to be enhanced by the long-term straw returning. Highly abundant DSRB optimal taxonomic units (OTUs) were found to be affiliated with the Nitrospirae supercluster (including the uncultured DsrAB lineage WX, lineage 10, and an unknown clade with GU372064), the Environmental supercluster 1 (including the uncultured DsrAB lineage 8 and an unknown lineage related to EF065019), and the Firmicutes group (including the uncultured DsrAB lineage 6 and an unknown lineage between the uncultured DsrAB lineages 2 and 3). The environmental transformation from the upland to the paddy stages led to a decrease in the α-diversity and the number of detectable OTUs. The abundance of a few dominant DSRB OTUs was changed by either the environmental transformation from the upland to the paddy stages or the effects caused by the long-term straw returning. In this study, the changes in the DSRB community structure correlated with the decrease in soil pH and total phosphorus content, and the increase in available sulfate and moisture contents.
The ubiquitous bacterial second messenger c‐di‐GMP is synthesized by diguanylate cyclase and degraded by c‐di‐GMP‐specific phosphodiesterase. The genome of Pseudomonas putida contains dozens of genes ...encoding diguanylate cyclase/phosphodiesterase, but the phenotypical–genotypical correlation and functional mechanism of these genes are largely unknown. Herein, we characterize the function and mechanism of a P. putida phosphodiesterase named DibA. DibA consists of a PAS domain, a GGDEF domain, and an EAL domain. The EAL domain is active and confers DibA phosphodiesterase activity. The GGDEF domain is inactive, but it promotes the phosphodiesterase activity of the EAL domain via binding GTP. Regarding phenotypic regulation, DibA modulates the cell surface adhesin LapA level in a c‐di‐GMP receptor LapD‐dependent manner, thereby inhibiting biofilm formation. Moreover, DibA interacts and colocalizes with LapD in the cell membrane, and the interaction between DibA and LapD promotes the PDE activity of DibA. Besides, except for interacting with DibA and LapD itself, LapD is found to interact with 11 different potential diguanylate cyclases/phosphodiesterases in P. putida, including the conserved phosphodiesterase BifA. Overall, our findings demonstrate the functional mechanism by which DibA regulates biofilm formation and expand the understanding of the LapD‐mediated c‐di‐GMP signaling network in P. putida.
The phosphodiesterase DibA interacts with the c‐di‐GMP receptor LapD to regulate the content of LapA on the cell surface, thereby inhibiting the biofilm formation of Psudomonas putida. The results of this study deepen our understanding of the specific regulation of biofilm by c‐di‐GMP signal transduction.
•Straw input and rice-growing stimulate soil nitrite oxidizing potential (NO).•Soil NO is related to a shift in Nitrospira-like NOB community structure.•Nitrospira community shift was significantly ...affected by pH, NH4+ and moisture.•Nitrobacter-like NOB was just not significantly affected in this system.•Nitrospira are more sensitive to straw input and rice-growing than Nitrobacter.
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Nitrite oxidation is recognized as an essential process of biogeochemical nitrogen cycling in agricultural ecosystems. How nitrite-oxidizing bacteria (NOB) respond to land managements (the effect from the long-term straw incorporation and environmental variability caused by the shift from the upland stage to the paddy stage) in a rapeseed-rice rotation field remains unclear. We found the nitrite oxidation (NO) in soils increased from the upland stage to the paddy stage. An inhibitory effect of the long-term straw incorporation on NO was detectable in the upland stage. The abundance of Nitrospira was always greater than Nitrobacter, and it was affected by the rice-growing and straw incorporation while Nitrobacter was not. NO correlated positively with the abundance of Nitrospira and with soluble sulfate (SO42−), soil moisture, pH and NH4+. The high-throughput sequencing analysis of the nitrite oxidoreductase nxrA and nxrB genes for Nitrobacter- and Nitrospira-like NOB was performed respectively. The dominating (relative abundance>1%) operational taxonomic units (OTUs) from Nitrobacter were closely related to Nitrobacter hamburgensis, whereas those from Nitrospira were affiliated with or related to lineage II, lineage V and several unknown groups. Heatmap analysis showed that a few dominant Nitrobacter OTUs were affected by the straw treatment or the rice-growing, and half of the dominant Nitrospira ones were explained by at least one of the variables. Multi-response permutation procedure (MRPP) and redundancy analyses showed that the Nitrospira-like NOB community changes were significantly shaped by the land managements and the soil chemical properties, including pH, moisture and NH4+, whereas that of the Nitrobacter-like NOB community was not. These results suggested that Nitrospira are more sensitive than Nitrobacter to land management in acid and fertilized soils of a rapeseed-rice rotation field trial.
Abstract
The ubiquitous bacterial second messenger cyclic diguanylate (c-di-GMP) coordinates diverse cellular processes through its downstream receptors. However, whether c-di-GMP participates in ...regulating nitrate assimilation is unclear. Here, we found that NasT, an antiterminator involved in nitrate assimilation in Pseudomonas putida, specifically bound c-di-GMP. NasT was essential for expressing the nirBD operon encoding nitrite reductase during nitrate assimilation. High-level c-di-GMP inhibited the binding of NasT to the leading RNA of nirBD operon (NalA), thus attenuating the antitermination function of NasT, resulting in decreased nirBD expression and nitrite reductase activity, which in turn led to increased nitrite accumulation in cells and its export. Molecular docking and point mutation assays revealed five residues in NasT (R70, Q72, D123, K127 and R140) involved in c-di-GMP-binding, of which R140 was essential for both c-di-GMP-binding and NalA-binding. Three diguanylate cyclases (c-di-GMP synthetases) were found to interact with NasT and inhibited nirBD expression, including WspR, PP_2557, and PP_4405. Besides, the c-di-GMP-binding ability of NasT was conserved in the other three representative Pseudomonas species, including P. aeruginosa, P. fluorescens and P. syringae. Our findings provide new insights into nitrate assimilation regulation by revealing the mechanism by which c-di-GMP inhibits nitrate assimilation via NasT.
Graphical Abstract
Graphical Abstract
The global regulatory molecule (p)ppGpp is synthesized under limited nutrition conditions and involves in many cellular processes in bacteria. (p)ppGpp has been reported to affect biofilm formation ...in several bacterial species. Here, we found that deletion of (p)ppGpp synthase genes of Pseudomonas putida KT2440 led to enhanced biofilm formation in polystyrene microtitre plates. Besides, the pellicle of this mutant formed at the air-liquid interface lost the robust structure and became frail. The biofilm formation and its structure are mainly determined by exopolysaccharides (EPSs) and adhesins. Transcriptional analysis of four EPS operons designated as pea, peb, alg and bcs and two adhesin genes nominated as lapA and lapF showed that the deletion of (p)ppGpp synthase genes increased the expression of peb, bcs and lapA but repressed the expression of pea and lapF. Furthermore, expression of the regulation factor FleQ was significantly augmented in (p)ppGpp-synthase mutants while the expression of sigma factor RpoS was reduced. Since FleQ and RpoS play important roles in regulating expression of EPS and adhesin genes, (p)ppGpp may mediate the synthesis of biofilm matrix via influencing these regulators to control the biofilm formation and pellicle structure.
Summary
Two‐component systems (TCSs) are predominant means by which bacteria sense and respond to environment signals. Genome of Pseudomonas putida contains dozens of putative TCS‐encoding genes, but ...phenotypical–genotypical correlation and transcriptional regulation of these genes are largely unknown. Herein, we characterized function and transcriptional regulation of a conserved P. putida TCS, named TarR–TarS. TarS (PP_0769) encodes a potential histidine kinase, and tarR (PP_0768) encodes a potential response regulator. Protein–protein interaction assay and phosphorylation assay confirmed that TarR–TarS was a functional TCS. Growth assay under antibiotics revealed that TarR–TarS positively regulated bacterial resistance to multiple antibiotics. Pull‐down assay revealed that TarR directly interacted with PP_0800 (a hypothetical protein) and GroEL (the chaperonin). GroEL played a positive role in antibiotic resistance, while PP_0800 seemed to have no effect on antibiotic resistance. The regulator FleQ indirectly activated tarR–tarS transcription. However, the second messenger c‐di‐GMP antagonized FleQ activation to inhibit tarR–tarS transcription. The sigma factor FliA directly activated tarR–tarS transcription via a consensus motif. These findings reveal function and transcriptional regulation of TarR–TarS, and enrich knowledge regarding the relationship between c‐di‐GMP and antibiotic susceptibility in P. putida.
Summary
Type VI secretion systems (T6SS) are specific antibacterial weapons employed by diverse bacteria to protect themselves from competitors. Pseudomonas putida KT2440 possesses a functional T6SS ...(K1‐T6SS) and exhibits antibacterial activity towards a broad range of bacteria. Here we found that the Wsp signal transduction system regulated K1‐T6SS expression via synthesizing the second messenger cyclic di‐GMP (c‐di‐GMP), thus mediating antibacterial activity in P. putida. High‐level c‐di‐GMP produced by Wsp system repressed the transcription of K1‐T6SS genes in structural operon and vgrG1 operon. Transcriptional regulator FleQ and ATPase FleN functioned as repressors in the Wsp system‐modulated K1‐T6SS transcription. However, FleQ and FleN functioned as activators in biofilm formation, and Wsp system promoted biofilm formation largely in a FleQ/FleN‐dependent manner. Furthermore, FleQ‐FleN complex bound directly to the promoter of K1‐T6SS structural operon in vitro, and c‐di‐GMP promoted the binding. Besides, P. putida biofilm cells showed higher c‐di‐GMP levels and lower antibacterial activity than planktonic cells. Overall, our findings reveal a mechanism by which Wsp system oppositely modulates antibacterial activity and biofilm formation via FleQ‐FleN, and demonstrate the relationship between plankton/biofilm lifestyles and antibacterial activity in P. putida.
Summary
The ubiquitous bacterial second messenger c‐di‐GMP is synthesized by diguanylate cyclase (DGC) and degraded by phosphodiesterase (PDE). Pseudomonas putida has dozens of DGC/PDE‐encoding genes ...in its genome, but the phenotypical–genotypical correlation and transcriptional regulation of these genes are largely unknown. Herein, we characterize function and transcriptional regulation of a P. putida c‐di‐GMP‐metabolizing enzyme, GcsA. GcsA consists of two per‐ARNT‐sim (PAS) domains, followed by a canonical conserved central sequence pattern (GGDEF) domain and a truncated EAL domain. In vitro analysis confirmed the DGC activity of GcsA. The phenotypic observation revealed that GcsA inhibited swimming motility in an FlgZ‐dependent manner. In terms of transcriptional regulation, gcsA was found to be cooperatively regulated by c‐di‐GMP and cAMP via their effectors, FleQ and Crp respectively. The transcription of gcsA was promoted by c‐di‐GMP and inhibited by cAMP. In vitro binding analysis revealed that FleQ indirectly regulated the transcription of gcsA, while Crp directly regulated the transcription of gcsA by binding to its promoter. Besides, an inverse relationship between the cellular c‐di‐GMP and cAMP levels in P. putida was confirmed. These findings provide basic knowledge regarding the function and transcriptional regulation of GcsA and demonstrate a crosstalk between c‐di‐GMP and cAMP in the regulation of the expression of GcsA in P. putida.
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