Complete genome sequence provides insights into the quorum sensing-related spoilage potential of Shewanella baltica 128 isolated from spoiled shrimp

Shewanella baltica 128 is a specific spoilage organism (SSO) isolated from the refrigerated shrimp that results in shrimp spoilage. This study reported the complete genome sequencing of this strain, with the primary annotations associated with amino acid transport and metabolism (8.66%), indicating that S. baltica 128 has good potential for degrading proteins. In vitro experiments revealed Shewanella baltica 128 could adapt to the stress conditions by regulating its growth and biofilm formation. Genes that related to the spoilage-related metabolic pathways, including trimethylamine metabolism (torT), sulfur metabolism (cysM), putrescine metabolism (speC), biofilm formation (rpoS) and serine protease production (degS), were identified. Genes (LuxS, pfs, LuxR and qseC) that related to the specific QS system were also identified. Complete genome sequence of S. baltica 128 provide insights into the QS-related spoilage potential, which might provide novel information for the development of new approaches for spoilage detection and prevention based on QS target.     

Involvement of the global regulators GrrS,RpoS, and SplIR in formation of biofilms in <Emphasis Type="Italic">Serratia plymuthica</Emphasis>

Most bacteria exist in the natural environment as biofilms, multicellular communities attached to various surfaces. Biofilms have a characteristic architecture and are enclosed in the exopolymer matrix. Bacterial cells in biofilms are extremely resistant to antibacterial factors. It was shown in this work that the GrrA/GrrS system of global regulators of gene expression and the sigma S subunit of RNA polymerase (RpoS) play a significant role in positive regulation of biofilm formation in the rhizospheric bacterium Serratia plymuthica IC1270. Inactivation of grrS and rpoS genes resulted in an up to six-to-sevenfold and four-to-fivefold reduction in biofilm formation, respectively. Mutation in the grrS gene decreased the capacity of the bacterium for swarming motility. The splIR Quorum Sensing (QS) system was shown to negatively influence the biofilm formation. Transfer of the recombinant plasmid containing cloned genes splI/splR of S. plymuthica HRO-C48 into S. plymuthica IC1270 cells led to a twofold decrease of their ability to form biofilms. Inactivation of the splI gene coding for the synthase of N-acyl-homoserine lactones in S. plymuthica HRO-C48 resulted in a 2–2.5-fold increase in the level of biofilm formation, whereas the transfer of plasmid carrying the cloned splI/splR genes into these mutant cells restored the biofilm formation to the normal level. The results obtained demonstrate that the formation of biofilms in S. plymuthica is positively regulated by the GrrA/GrrS and RpoS global regulators and is negatively regulated by the SplIR QS system.     

Characterization of <Emphasis Type="Italic">Edwardsiella tarda rpoS</Emphasis>: effect on serum resistance,chondroitinase activity,biofilm formation,and autoinducer synthetases expression

In this study, rpoS gene was identified from Edwardsiella tarda EIB202 and its functional role was analyzed by using an in-frame deletion mutant ∆rpoS and the complemental strain rpoS ⁺. Compared with the wild type and rpoS ⁺, ∆rpoS was impaired in terms of the ability to survive under oxidative stress and nutrient starvation, as well as the resistance to 50% serum of Scophthalmus maximus in 3 h, demonstrating essential roles of RpoS in stress adaptation. The rpoS mutant also displayed markedly increased chondroitinase activity and biofilm formation. Real-time polymerase chain reaction revealed that the expression level of quorum sensing autoinducer synthetase genes luxS and edwI was increased by 3.7- and 2.5-fold in the rpoS mutant strain. Those results suggested that rpoS might be involved in the negative or positive regulation of chondroitinase and biofilm formation, or quorum sensing networks in E. tarda, respectively. Although there were no obvious differences between the wild-type and the rpoS mutant in adherence of epithelioma papulosum cyprini (EPC) cell and in the lethality on fish model, rpoS deletion leads to the drastically reduced capacity for E. tarda to internalize in EPC cells, indicating that RpoS was, while not the main, the factor required for the virulence network of E. tarda.     

基于比较基因组学分析大黄鱼来源波罗的海希瓦氏菌的防御系统

【目的】波罗的海希瓦氏菌是冷藏海产品中常见的腐败菌,通过全基因组测序和转录组测序,分析它们的规律成簇间隔短回文重复序列(clustered regularly interspaced short palindromic repeats,CRISPR)系统和限制修饰(restricted modification,R-M)系统,为波罗的海希瓦氏菌的基础生物学研究和海产品中微生物的致腐机制提供理论基础。【方法】分析大黄鱼来源波罗的海希瓦氏菌SB-19株和W-3株的致腐能力,对W-3株的全基因组序列进行测序、组装和注释,结合已报道的SB-19株和27株希瓦氏菌的全基因组序列,采用比较基因组学方法探究它们的CRISPR和R-M系统的差异,进而对SB-19株和W-3株在不同生长时期进行转录组测序,以及两株菌内致腐相关基因的共进化分析。【结果】灭菌大黄鱼汁中产生挥发性盐基总氮和三甲胺值显示波罗的海希瓦氏菌SB-19株和W-3株分别为强致腐能力和弱致腐能力菌株;平均核苷酸一致性证实SB-19株和W-3株为波罗的海希瓦氏菌,但基于全基因组构建的系统发育树则发现二者之间存在遗传信息上的差异;SB-19株...     

Role of RpoS in stress survival,synthesis of extracellular autoinducer 2, and virulence in <Emphasis Type="Italic">Vibrio alginolyticus</Emphasis>

Vibrio alginolyticus, a marine bacterium, is an opportunistic pathogen capable of causing vibriosis with high mortality to fishes in the South China Sea. Stress resistance is very important for its survival in the natural environment and upon infection of the host. RpoS, an alternative sigma factor, is considered as an important regulator involved in stress response and virulence in many pathogens. In this study, the rpoS gene was cloned and characterized to evaluate the role of RpoS in V. alginolyticus. The predicted protein showed high identity with other reported rpoS gene products. The in-frame deleted mutation of rpoS in V. alginolyticus led to sensitivity of the strain to ethanol, hyperosmolarity, heat, and hydrogen peroxide challenges. Further studies showed that extracellular autoinducer 2 level, four of seven detected protease activities, and cytotoxicity of extracellular products were markedly decreased in the rpoS mutant compared with that in the wild-type strain. The results indicated that the global regulator RpoS was part of the regulatory networks of virulence and LuxS quorum sensing system.     

Role of rpoS in Escherichia coli O157:H7 Strain H32 Biofilm Development and Survival

The protein RpoS is responsible for mediating cell survival during the stationary phase by conferring cell resistance to various stressors and has been linked to biofilm formation. In this study, the role of the rpoS gene in Escherichia coli O157:H7 biofilm formation and survival in water was investigated. Confocal scanning laser microscopy of biofilms established on coverslips revealed a nutrient-dependent role of rpoS in biofilm formation, where the biofilm biomass volume of the rpoS mutant was 2.4- to 7.5-fold the size of its rpoS⁺ wild-type counterpart in minimal growth medium. The enhanced biofilm formation of the rpoS mutant did not, however, translate to increased survival in sterile double-distilled water (ddH₂O), filter-sterilized lake water, or unfiltered lake water. The rpoS mutant had an overall reduction of 3.10 and 5.30 log₁₀ in sterile ddH₂O and filter-sterilized lake water, respectively, while only minor reductions of 0.53 and 0.61 log₁₀ in viable counts were observed for the wild-type form in the two media over a 13-day period, respectively. However, the survival rates of the detached biofilm-derived rpoS⁺ and rpoS mutant cells were comparable. Under the competitive stress conditions of unfiltered lake water, the advantage conferred by the presence of rpoS was lost, and both the wild-type and knockout forms displayed similar declines in viable counts. These results suggest that rpoS does have an influence on both biofilm formation and survival of E. coli O157:H7 and that the advantage conferred by rpoS is contingent on the environmental conditions.     

Kinetics of biofilm formation and desiccation survival of Listeria monocytogenes in single and dual species biofilms with Pseudomonas fluorescens,Serratia proteamaculans or Shewanella baltica on food-grade stainless steel surfaces

This study investigated the dynamics of static biofilm formation (100% RH, 15 °C, 48–72 h) and desiccation survival (43% RH, 15 °C, 21 days) of Listeria monocytogenes, in dual species biofilms with the common spoilage bacteria, Pseudomonas fluorescens, Serratia proteamaculans and Shewanella baltica, on the surface of food grade stainless steel. The Gram-negative bacteria reduced the maximum biofilm population of L. monocytogenes in dual species biofilms and increased its inactivation during desiccation. However, due to the higher desiccation resistance of Listeria relative to P. fluorescens and S. baltica, the pathogen survived in greater final numbers. In contrast, S. proteamaculans outcompeted the pathogen during the biofilm formation and exhibited similar desiccation survival, causing the N_{21 days} of Serratia to be ca 3 Log₁₀(CFU cm^?2) greater than that of Listeria in the dual species biofilm. Microscopy revealed biofilm morphologies with variable amounts of exopolymeric substance and the presence of separate microcolonies. Under these simulated food plant conditions, the fate of L. monocytogenes during formation of mixed biofilms and desiccation depended on the implicit characteristics of the co-cultured bacterium.     

<Emphasis Type="Italic">rpoS</Emphasis> Gene Expression in Carbon-Starved Cultures of the Polyhydroxyalkanoate-Accumulating Species <Emphasis Type="Italic">Pseudomonas oleovorans</Emphasis>

The expression of the rpoS gene during PHA depolymerization was monitored in Pseudomonas oleovorans GPo1 and its mutant defective in PHA degradation by analyzing the tolerance to oxidative and thermal stresses and the RpoS intracellular content. An increase in the tolerance to H₂O₂ and heat shock was observed coincidentally with PHA degradation. Western blotting experiments performed in carbon-starved cultures showed that the RpoS levels were higher in the wild type than in the mutant strain. Complementation of the phaZ mutation restores the wild-type RpoS levels. These results suggest a probable association between PHA depolymerization and the stress tolerance phenotype controlled by RpoS.     

RpoS and Indole Signaling Control the Virulence of Vibrio anguillarum towards Gnotobiotic Sea Bass (Dicentrarchus labrax) Larvae

Quorum sensing, bacterial cell-to-cell communication with small signal molecules, controls the virulence of many pathogens. In contrast to other vibrios, neither the VanI/VanR acylhomoserine lactone quorum sensing system, nor the three-channel quorum sensing system affects virulence of the economically important aquatic pathogen Vibrio anguillarum. Indole is another molecule that recently gained attention as a putative signal molecule. The data presented in this study indicate that indole signaling and the alternative sigma factor RpoS have a significant impact on the virulence of V. anguillarum. Deletion of rpoS resulted in increased expression of the indole biosynthesis gene tnaA and in increased production of indole. Both rpoS deletion and the addition of exogenous indole (50–100 µM) resulted in decreased biofilm formation, exopolysaccharide production (a phenotype that is required for pathogenicity) and expression of the exopolysaccharide synthesis gene wbfD. Further, indole inhibitors increased the virulence of the rpoS deletion mutant, suggesting that indole acts downstream of RpoS. Finally, in addition to the phenotypes found to be affected by indole, the rpoS deletion mutant also showed increased motility and decreased sensitivity to oxidative stress.     

The effect of the rpoSam allele on gene expression and stress resistance in Escherichia coli

The RNA polymerase associated with RpoS transcribes many genes related to stationary phase and stress survival in Escherichia coli. The DNA sequence of rpoS exhibits a high degree of polymorphism. A C to T transition at position 99 of the rpoS ORF, which results in a premature amber stop codon often found in E. coli strains. The rpoSam mutant expresses a truncated and partially functional RpoS protein. Here, we present new evidence regarding rpoS polymorphism in common laboratory E. coli strains. One out of the six tested strains carries the rpoSam allele, but expressed a full-length RpoS protein owing to the presence of an amber supressor mutation. The rpoSam allele was transferred to a non-suppressor background and tested for RpoS level, stress resistance and for the expression of RpoS and sigma70-dependent genes. Overall, the rpoSam strain displayed an intermediate phenotype regarding stress resistance and the expression of σ^S-dependent genes when compared to the wild-type rpoS ⁺ strain and to the rpoS null mutant. Surprisingly, overexpression of rpoSam had a differential effect on the expression of the σ⁷⁰-dependent genes phoA and lacZ that, respectively, encode the enzymes alkaline phosphatase and β-galactosidase. The former was enhanced while the latter was inhibited by high levels of RpoSam.     

Engineering Synthetic Multistress Tolerance in Escherichia coli by Using a Deinococcal Response Regulator,DR1558

Cellular robustness is an important trait for industrial microbes, because the microbial strains are exposed to a multitude of different stresses during industrial processes, such as fermentation. Thus, engineering robustness in an organism in order to push the strains toward maximizing yield has become a significant topic of research. We introduced the deinococcal response regulator DR1558 into Escherichia coli (strain Ec-1558), thereby conferring tolerance to hydrogen peroxide (H₂O₂). The reactive oxygen species (ROS) level in strain Ec-1558 was reduced due to the increased KatE catalase activity. Among four regulators of the oxidative-stress response, OxyR, RpoS, SoxS, and Fur, we found that the expression of rpoS increased in Ec-1558, and we confirmed this increase by Western blot analysis. Electrophoretic mobility shift assays showed that DR1558 bound to the rpoS promoter. Because the alternative sigma factor RpoS regulates various stress resistance-related genes, we performed stress survival analysis using an rpoS mutant strain. Ec-1558 was able to tolerate a low pH, a high temperature, and high NaCl concentrations in addition to H₂O₂, and the multistress tolerance phenotype disappeared in the absence of rpoS. Microarray analysis clearly showed that a variety of stress-responsive genes that are directly or indirectly controlled by RpoS were upregulated in strain Ec-1558. These findings, taken together, indicate that the multistress tolerance conferred by DR1558 is likely routed through RpoS. In the present study, we propose a novel strategy of employing an exogenous response regulator from polyextremophiles for strain improvement.     

Low-Shear Modeled Microgravity Alters the Salmonella enterica Serovar Typhimurium Stress Response in an RpoS-Independent Manner

We have previously demonstrated that low-shear modeled microgravity (low-shear MMG) serves to enhance the virulence of a bacterial pathogen, Salmonella enterica serovar Typhimurium. The Salmonella response to low-shear MMG involves a signaling pathway that we have termed the low-shear MMG stimulon, though the identities of the low-shear MMG stimulon genes and regulatory factors are not known. RpoS is the primary sigma factor required for the expression of genes that are induced upon exposure to different environmental-stress signals and is essential for virulence in mice. Since low-shear MMG induces a Salmonella acid stress response and enhances Salmonella virulence, we reasoned that RpoS would be a likely regulator of the Salmonella low-shear MMG response. Our results demonstrate that low-shear MMG provides cross-resistance to several environmental stresses in both wild-type and isogenic rpoS mutant strains. Growth under low-shear MMG decreased the generation time of both strains in minimal medium and increased the ability of both strains to survive in J774 macrophages. Using DNA microarray analysis, we found no evidence of induction of the RpoS regulon by low-shear MMG but did find that other genes were altered in expression under these conditions in both the wild-type and rpoS mutant strains. Our results indicate that, under the conditions of these studies, RpoS is not required for transmission of the signal that induces the low-shear MMG stimulon. Moreover, our studies also indicate that low-shear MMG can be added to a short list of growth conditions that can serve to preadapt an rpoS mutant for resistance to multiple environmental stresses.     

Autoinduction of RpoS Biosynthesis in the Biocontrol Strain <Emphasis Type="Italic">Pseudomonas</Emphasis> sp. M18

The rpoS gene from Pseudomonas sp. M18, which encodes predicted protein (an alternative sigma factor s, σ^S, or σ³⁸) with 99.5% sequence identity with RpoS from Pseudomonas aeruginosa PAO1, was first cloned. In order to investigate the mechanism of rpoS expression, an rpoS null mutant, named M18S, was constructed with insertion of aacC1 cassette bearing a gentamycin resistance gene. With introduction of a plasmid containing an rpoS′–′lacZ translational fusion (pMERS) to wild-type strain M18 or M18S, it was first found that β-galactosidase activity expressed in strain M18S (pMERS) decreased to fourfold of that expressed in the strain M18 (pMERS). When strain M18S (pMERS) was introduced with another plasmid pBBS containing the wild-type rpoS gene, its β-galactosidase expression level was enhanced and almost restored to that in strain M18 (pMERS). Similarly, expression of β-galactosidase from a chromosomal fusion of the promoter of the wild-type rpoS gene with lacZ (rpoS–lacZ) was enhanced fivefold in the presence of a plasmid with the wild-type rpoS gene. With these findings, it is suggested that RpoS sigma factor may be involved in autoinducing its own gene expression in Pseudomonas sp. M18.     

海产品嗜冷菌波罗的海希瓦氏菌中3个冷激蛋白功能分析

【目的】波罗的海希瓦氏菌是冷藏海产品中常见的腐败菌,而该菌中关于冷激蛋白的功能研究尚未见报道。本研究从分子生物学角度分析波罗的海希瓦氏菌中3个冷激蛋白各自的功能。【方法】采用BEAST软件分析γ-变形菌纲中部分食源性微生物的冷激蛋白进化时间,接着利用实时荧光定量PCR方法检测波罗的海希瓦氏菌3个冷激蛋白基因的表达规律,进而构建3个冷激蛋白的基因敲除株,分析敲除株在不同温度和不同环境胁迫条件下的生长状况、群体感应现象以及致腐能力,最后构建3个冷激蛋白的异源表达菌株并分析它们在不同温度和不同环境胁迫条件下的生长状况。【结果】波罗的海希瓦氏菌中鉴定到3个冷激蛋白,分别为cspC、cspD、cspG。所有γ-变形菌纲的cspD基因单独聚成一支,并于1 109.6百万年前与其他csp基因相分离,波罗的海希瓦氏菌的cspC和cspG在858.8百万年前互相分开。cspG基因是波罗的海希瓦氏菌低温生存的必需基因,且广泛响应环境胁迫条件;cspC基因对cspG基因功能的实施起辅助作用;cspD不响应冷激,但却会随生长阶段的变化而发生变化。此外,cspC基因和cspG基因在低温条件下与细菌的致腐能力相关。【结论】波罗的海希瓦氏菌3个冷激蛋白基因各有不同,且cspC基因和cspG基因与该菌致腐能力有关,这为今后研究腐败菌的冷适应和致腐机制提供了新思路。     

Contribution of quorum‐sensing system to hexadecane degradation and biofilm formation in Acinetobacter sp. strain DR1

Aims: To investigate roles of quorum‐sensing (QS) system in Acinetobacter sp. strain DR1 and rifampicin‐resistant variant (hereinafter DR1R). Methods and Results: The DR1 strain generated three putative acyl homoserine lactones (AHLs), while the DR1R produced only one signal and QS signal production was abrogated in the aqsI (LuxI homolog) mutant. The hexadecane‐degradation and biofilm‐formation capabilities of DR1, DR1R, and aqsI mutants were compared, along with their proteomic data. Proteomics analysis revealed that the AHL lactonase responsible for degrading QS signal was highly upregulated in both DR1R and aqsI mutant, also showed that several proteins, including ppGpp synthase, histidine kinase sensors, might be under the control of QS signalling. Interestingly, biofilm‐formation and hexadecane‐biodegradation abilities were reduced more profoundly in the aqsI mutant. These altered phenotypes of the aqsI mutant were restored via the addition of free wild‐type cell supernatant and exogenous C₁₂‐AHL. Conclusions: The QS system in strain DR1 contributes to hexadecane degradation and biofilm formation. Significance and Impact of the Study: This is the first report to demonstrate that a specific QS signal appears to be a critical factor for hexadecane degradation and biofilm formation in Acinetobacter sp. strain DR1.