Outer membrane defect and stronger biofilm formation caused by inactivation of a gene encoding for heptosyltransferase I in Cronobacter sakazakii ATCC BAA-894

Aims: To investigate the role of lipopolysaccharide (LPS) structure in the stability of outer membrane and the ability of biofilm formation in Cronobacter sakazakii. Methods and Results: A C. sakazakii mutant strain LWW02 was constructed by inactivating the gene ESA_04107 encoding for heptosyltransferase I. LPS were purified from LWW02, and changes in their structure were confirmed by thin‐layer chromatography and electrospray ionization mass spectrometry. Comparing with the wild‐type strain BAA‐894, slower growth, higher membrane permeability, higher surface hydrophobicity, stronger ability of autoaggregation and biofilm formation were observed for the mutant strain LWW02. Conclusions: The gene ESA_04107 encodes heptosyltransferase I in C. sakazakii ATCC BAA‐894. The cleavage of LPS in C. sakazakii could cause its outer membrane defects and increase its ability to form biofilms. Significance and Impact of the Study: The study is important for understanding the pathogenic mechanism and efficient control of C. sakazakii.     

The novel virulence-related gene nlxA in the lipopolysaccharide cluster of Xanthomonas citri ssp. citri is involved in the production of lipopolysaccharide and extracellular polysaccharide, motility, biofilm formation and stress resistance

Lipopolysaccharide (LPS) is an important virulence factor of Xanthomonas citri ssp. citri, the causative agent of citrus canker disease. In this research, a novel gene, designated as nlxA (novel LPS cluster gene of X. citri ssp. citri), in the LPS cluster of X. citri ssp. citri 306, was characterized. Our results indicate that nlxA is required for O‐polysaccharide biosynthesis by encoding a putative rhamnosyltransferase. This is supported by several lines of evidence: (i) NlxA shares 40.14% identity with WsaF, which acts as a rhamnosyltransferase; (ii) sodium dodecylsulphate‐polyacrylamide gel electrophoresis analysis showed that four bands of the O‐antigen part of LPS were missing in the LPS production of the nlxA mutant; this is also consistent with a previous report that the O‐antigen moiety of LPS of X. citri ssp. citri is composed of a rhamnose homo‐oligosaccharide; (iii) mutation of nlxA resulted in a significant reduction in the resistance of X. citri ssp. citri to different stresses, including sodium dodecylsulphate, polymyxin B, H₂O₂, phenol, CuSO₄ and ZnSO₄. In addition, our results indicate that nlxA plays an important role in extracellular polysaccharide production, biofilm formation, stress resistance, motility on semi‐solid plates, virulence and in planta growth in the host plant grapefruit.     

Phage‐selected lipopolysaccharide mutants of Pectobacterium atrosepticum exhibit different impacts on virulence

Aims: To positively select Pectobacterium atrosepticum (Pa) mutants with cell surface defects and to assess the impact of these mutations on phytopathogenesis. Methods and Results: Several phages were isolated from treated sewage effluent and were found to require bacterial lipopolysaccharide (LPS) for infection. Two strains with distinct mutations in LPS were obtained by transposon mutagenesis. Along with a third LPS mutant, these strains were characterized with respect to various virulence‐associated phenotypes, including growth rate, motility and exoenzyme production, demonstrating that LPS mutations are pleiotropic. Two of the strains were deficient in the synthesis of the O‐antigen portion of LPS, and both were less virulent than the wild type. A waaJ mutant, which has severe defects in LPS biosynthesis, was dramatically impaired in potato tuber rot assays. The infectivity of these novel phages on 32 additional strains of Pa was tested, showing that most Pa isolates were sensitive to the LPS‐dependent phages. Conclusions: Native LPS is crucial for optimal growth, survival and virulence of Pa in vivo, but simultaneously renders such strains susceptible to phage infection. Significance and Impact of the Study: This work demonstrates the power of phages to select and identify the virulence determinants on the bacterial surface, and as potential biocontrol agents for Pa infections.     

Identification of the lipopolysaccharide O‐antigen biosynthesis priming enzyme and the O‐antigen ligase in Myxococcus xanthus: critical role of LPS O‐antigen in motility and development

Myxococcus xanthus is a model bacterium to study social behavior. At the cellular level, the different social behaviors of M. xanthus involve extensive cell–cell contacts. Here, we used bioinformatics, genetics, heterologous expression and biochemical experiments to identify and characterize the key enzymes in M. xanthus implicated in O‐antigen and lipopolysaccharide (LPS) biosynthesis and examined the role of LPS O‐antigen in M. xanthus social behaviors. We identified WbaP_Mx (MXAN_2922) as the polyisoprenyl‐phosphate hexose‐1‐phosphate transferase responsible for priming O‐antigen synthesis. In heterologous expression experiments, WbaP_Mx complemented a Salmonella enterica mutant lacking the endogenous WbaP that primes O‐antigen synthesis, indicating that WbaP_Mx transfers galactose‐1‐P to undecaprenyl‐phosphate. We also identified WaaL_Mx (MXAN_2919), as the O‐antigen ligase that joins O‐antigen to lipid A‐core. Our data also support the previous suggestion that Wzm_Mx (MXAN_4622) and Wzt_Mx (MXAN_4623) form the Wzm/Wzt ABC transporter. We show that mutations that block different steps in LPS O‐antigen synthesis can cause pleiotropic phenotypes. Also, using a wbaP_Mx deletion mutant, we revisited the role of LPS O‐antigen and demonstrate that it is important for gliding motility, conditionally important for type IV pili‐dependent motility and required to complete the developmental program leading to the formation of spore‐filled fruiting bodies.     

A spider web strategy of type IV pili‐mediated migration to build a fibre‐like Psl polysaccharide matrix in Pseudomonas aeruginosa biofilms

Bacterial motilities participate in biofilm development. However, it is unknown how/if bacterial motility affects formation of the biofilm matrix. Psl polysaccharide is a key biofilm matrix component of Pseudomonas aeruginosa. Here we report that type IV pili (T4P)‐mediated bacterial migration leads to the formation of a fibre‐like Psl matrix. Deletion of T4P in wild type and flagella‐deficient strains results in loss of the Psl‐fibres and reduction of biofilm biomass in flow cell biofilms as well as pellicles at air‐liquid interface. Bacteria lacking T4P‐driven twitching motility including those that still express surface T4P are unable to form the Psl‐fibres. Formation of a Psl‐fibre matrix is critical for efficient biofilm formation, yet does not require flagella and polysaccharide Pel or alginate. The Psl‐fibres are likely formed by Psl released from bacteria during T4P‐mediated migration, a strategy similar to spider web formation. Starvation can couple Psl release and T4P‐driven twitching motility. Furthermore, a radial‐pattern Psl‐fibre matrix is present in the middle of biofilms, a nutrient‐deprived region. These imply a plausible model for how bacteria respond to nutrient‐limited local environment to build a polysaccharide‐fibre matrix by T4P‐dependent bacterial migration strategy. This strategy may have general significance for bacterial survival in natural and clinical settings.     

Motility and chemotaxis in Agrobacterium tumefaciens surface attachment and biofilm formation

Bacterial motility mechanisms, including swimming, swarming, and twitching, are known to have important roles in biofilm formation, including colonization and the subsequent expansion into mature structured surface communities. Directed motility requires chemotaxis functions that are conserved among many bacterial species. The biofilm-forming plant pathogen Agrobacterium tumefaciens drives swimming motility by utilizing a small group of flagella localized to a single pole or the subpolar region of the cell. There is no evidence for twitching or swarming motility in A. tumefaciens. Site-specific deletion mutations that resulted in either aflagellate, flagellated but nonmotile, or flagellated but nonchemotactic A. tumefaciens derivatives were examined for biofilm formation under static and flowing conditions. Nonmotile mutants were significantly deficient in biofilm formation under static conditions. Under flowing conditions, however, the aflagellate mutant rapidly formed aberrantly dense, tall biofilms. In contrast, a nonmotile mutant with unpowered flagella was clearly debilitated for biofilm formation relative to the wild type. A nontumbling chemotaxis mutant was only weakly affected with regard to biofilm formation under nonflowing conditions but was notably compromised in flow, generating less adherent biomass than the wild type, with a more dispersed cellular arrangement. Extragenic suppressor mutants of the chemotaxis-impaired, straight-swimming phenotype were readily isolated from motility agar plates. These mutants regained tumbling at a frequency similar to that of the wild type. Despite this phenotype, biofilm formation by the suppressor mutants in static cultures was significantly deficient. Under flowing conditions, a representative suppressor mutant manifested a phenotype similar to yet distinct from that of its nonchemotactic parent.     

Efficient rhizosphere colonization by Pseudomonas fluorescens f113 mutants unable to form biofilms on abiotic surfaces

Motility is a key trait for rhizosphere colonization by Pseudomonas fluorescens. Mutants with reduced motility are poor competitors, and hypermotile, more competitive phenotypic variants are selected in the rhizosphere. Flagellar motility is a feature associated to planktonic, free‐living single cells, and although it is necessary for the initial steps of biofilm formation, bacteria in biofilm lack flagella. To test the correlation between biofilm formation and rhizosphere colonization, we have used P. fluorescens F113 hypermotile derivatives and mutants affected in regulatory genes which in other bacteria modulate biofilm development, namely gacS (G), sadB (S) and wspR (W). Mutants affected in these three genes and a hypermotile variant (V35) isolated from the rhizosphere were impaired in biofilm formation on abiotic surfaces, but colonized the alfalfa root apex as efficiently as the wild‐type strain, indicating that biofilm formation on abiotic surfaces and rhizosphere colonization follow different regulatory pathways in P. fluorescens. Furthermore, a triple mutant gacSsadBwspR (GSW) and V35 were more competitive than the wild‐type strain for root‐tip colonization, suggesting that motility is more relevant in this environment than the ability to form biofilms on abiotic surfaces. Microscopy showed the same root colonization pattern for P. fluorescens F113 and all the derivatives: extensive microcolonies, apparently held to the rhizoplane by a mucigel that seems to be plant produced. Therefore, the ability to form biofilms on abiotic surfaces does not necessarily correlates with efficient rhizosphere colonization or competitive colonization.     

Involvement of the MyD88‐independent pathway in controlling the intracellular fate of Burkholderia pseudomallei infection in the mouse macrophage cell line RAW 264.7

Burkholderia pseudomallei is a facultative intracellular Gram‐negative bacterium which is capable of surviving and multiplying inside macrophages. B. pseudomallei strain SRM117, a LPS mutant which lacks the O‐antigenic polysaccharide moiety, is more susceptible to macrophage killing during the early phase of infection than is its parental wild type strain (1026b). In this study, it was shown that the wild type is able to induce expression of genes downstream of the MyD88‐dependent (iκbζ, il‐6 and tnf‐α), but not of the MyD88‐independent (inos, ifn‐β and irg‐1), pathways in the mouse macrophage cell line RAW 264.7. In contrast, LPS mutant‐infected macrophages were able to express genes downstream of both pathways. To elucidate the significance of activation of the MyD88‐independent pathway in B. pseudomallei‐infected macrophages, the expression of TBK1, an essential protein in the MyD88‐independent pathway, was silenced prior to the infection. The results showed that silencing the tbk1 expression interferes with the gene expression profile in LPS mutant‐infected macrophages and allows the bacteria to replicate intracellularly, thus suggesting that the MyD88‐independent pathway plays an essential role in controlling intracellular survival of the LPS mutant. Moreover, exogenous IFN‐γ upregulated gene expression downstream of the MyD88‐independent pathway, and interfered with intracellular survival in both wild type and tbk1‐knockdown macrophages infected with either the wild type or the LPS mutant. These results suggest that gene expression downstream of the MyD88‐independent pathway is essential in regulating the intracellular fate of B. pseudomallei, and that IFN‐γ regulates gene expression through the TBK1‐independent pathway.     

Characterization of starvation‐induced dispersion in Pseudomonas putida biofilms: genetic elements and molecular mechanisms

Pseudomonas putida OUS82 biofilm dispersal was previously shown to be dependent on the gene PP0164 (here designated lapG). Sequence and structural analysis has suggested that the LapG geneproduct belongs to a family of cysteine proteinases that function in the modification of bacterial surface proteins. We provide evidence that LapG is involved in P. putida OUS82 biofilm dispersal through modification of the outer membrane‐associated protein LapA. While the P. putida lapG mutant formed more biofilm than the wild‐type, P. putida lapA and P. putida lapAG mutants displayed decreased surface adhesion and were deficient in subsequent biofilm formation, suggesting that LapG affects LapA, and that the LapA protein functions both as a surface adhesin and as a biofilm matrix component. Lowering of the intracellular c‐di‐GMP level via induction of an EAL domain protein led to dispersal of P. putida wild‐type biofilm but did not disperse P. putida lapG biofilm, indicating that LapG exerts its activity on LapA in response to a decrease in the intracellular c‐di‐GMP level. In addition, evidence is provided that associated to LapA a cellulase‐degradable exopolysaccharide is part of the P. putida biofilm matrix.     

Cellular hydrophobicity of Listeria monocytogenes involves initial attachment and biofilm formation on the surface of polyvinyl chloride

Aims: To clarify the cellular properties of Listeria monocytogenes involved in adhesion to and biofilm formation on polyvinyl chloride, a widely used material in the food manufacturing process. Methods and Results: A significant correlation between the ability of initial adherence to and biofilm formation on PVC was observed for 24 L. monocytogenes strains (Spearman rank‐correlation coefficient, r_s = 0·89). The swimming motility assay revealed no relationship between initial adherence and motility of L. monocytogenes. The microbial adhesion to solvent assay revealed an interaction of L. monocytogenes cells with nonpolar solvents, and a significant correlation was also observed between the degree of interaction with nonpolar solvents and initial adherence to PVC (r_s = 0·87 and r_s = 0·84, between initial adherence and affinities to decane and hexadecane, respectively). Conclusions: Results indicate that cellular hydrophobicity of L. monocytogenes is an important property involved in the initial adherence to and biofilm formation on PVC. Significance and Impact of Study: This study clarified the factors involved in the adherence to and biofilm formation ability of L. monocytogenes strains with PVC.     

茎瘤固氮根瘤菌环二鸟苷酸代谢相关基因的功能鉴定

【目的】考察茎瘤固氮根瘤菌ORS571中c-di-GMP合成酶AZC-2412的编码基因缺失的突变表型,初步探究其功能机理。【方法】本实验构建基于cre-loxp重组酶系统的根瘤菌基因敲除系统,以及采用三亲接合技术构建突变株。测定野生型和突变株的生长速率、趋化能力、胞外多糖产量、生物膜形成等表型。【结果】突变株与野生型生长速率几乎相同。与野生型相比突变株由于细胞内c-di-GMP水平降低,胞外多糖、生物膜产量等均有所下降。【结论】实验表明,环二鸟苷酸合成酶AZC-2412缺失,使得c-di-GMP水平降低,对胞外多糖生成、细菌的运动能力、生物膜的形成、细胞絮凝、与植物的互作等均有调控作用。     

Anti-biofilm activity of Quercus infectoria G. Olivier against methicillin-resistant Staphylococcus aureus

Aims: To establish the effect of Quercus infectoria G. Olivier extract and its main constituent, tannic acid, on staphylococcal biofilm and their anti‐biofilm mechanisms. Methods and Results: Anti‐biofilm activity of the plant materials on clinical isolated of methicillin‐resistant Staphylococcus aureus and methicillin‐susceptible Staph. aureus was employed using a crystal violet‐stained microtiter plate method. The extract at minimum inhibitory concentration (MIC; 0·25 mg ml^?1) was significantly reduced the biofilm formation of the isolates (P < 0·05). The effect on staphylococcal cell surface hydrophobicity (CSH) of the test compounds was investigated as a possible mode of action of the anti‐biofilm activity. The hydrophobicity index of all the bacterial isolates increased following treatment with supra‐MIC, MIC and sub‐MIC of the extract and tannic acid. Observation of the treated bacterial cells by electron microscopy revealed that the test compounds caused clumps of partly divided cocci with thickened and slightly rough cell wall. Conclusions: The results indicated that Q. infectoria extract and tannic acid affected staphylococcal biofilm formation and their effect on bacterial CSH and cell wall may involve in the anti‐biofilm activity. Significance and Impact of the Study: This evidence highlighted the anti‐biofilm potency of the natural products and clarified their anti‐biofilm mechanisms.     

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.     

Flagella and Pili of Xanthomonas axonopodis pv. glycines are associated with motility,biofilm formation and virulence on soybean

Targeted mutations in flgK, and pilD genes in strain KU‐P‐SW005 of Xanthomonas axonopodis pv. glycines, the cause of pustule disease on soybean, led to altered motility phenotypes. The flgK mutants lacked a monopolar flagellum and lost swimming motility, whereas the pilD mutant lacked type IV pili and was unable to move via twitching, a form of surface motility not previously reported for this pathogen. The flgK and pilD mutants were also altered in biofilm production. The flgK and pilD mutants caused reduced disease in susceptible soybean cultivars Spencer when compared to KU‐P‐SW005. Cell counts of the flgK and pilD mutants on plants remained equivalent to KU‐P‐SW005 10 days after inoculation. Complementation of flgK and pilD mutants restored all phenotypes to wild‐type levels. Therefore, flgK and pilD genes that are required for swimming and twitching motility also affected biofilm formation and virulence on soybean.     

The Francisella O‐antigen mediates survival in the macrophage cytosol via autophagy avoidance

Autophagy is a key innate immune response to intracellular parasites that promotes their delivery to degradative lysosomes following detection in the cytosol or within damaged vacuoles. Like Listeria and Shigella, which use specific mechanisms to avoid autophagic detection and capture, the bacterial pathogen Francisella tularensis proliferates within the cytosol of macrophages without demonstrable control by autophagy. To examine how Francisella evades autophagy, we screened a library of F. tularensis subsp. tularensis Schu S4 HimarFT transposon mutants in GFP‐LC3‐expressing murine macrophages by microscopy for clones localized within autophagic vacuoles after phagosomal escape. Eleven clones showed autophagic capture at 6 h post‐infection, whose HimarFT insertions clustered to fourgenetic loci involved in lipopolysaccharidic and capsular O‐antigen biosynthesis. Consistent with the HimarFT mutants, in‐frame deletion mutants of two representative loci, FTT1236 and FTT1448c (manC), lacking both LPS and capsular O‐antigen, underwent phagosomal escape but were cleared from the host cytosol. Unlike wild‐type Francisella, the O‐antigen deletion mutants were ubiquitinated, and recruited the autophagy adaptor p62/SQSTM1 and LC3 prior to cytosolic clearance. Autophagy‐deficient macrophages partially supported replication of both mutants, indicating that O‐antigen‐lacking Francisella are controlled by autophagy. These data demonstrate the intracellular protective role of this bacterial surface polysaccharide against autophagy.     

Involvement of PorK,a component of the type IX secretion system,in Prevotella melaninogenica pathogenicity

Prevotella melaninogenica is a gram‐negative anaerobic commensal bacterium that resides in the human oral cavity and is isolated as a pathogen of suppurative diseases both inside and outside the mouth. However, little is known about the pathogenic factors of P. melaninogenica. The periodontal pathogens Porphyromonas gingivalis and Tanerella forsythia secrete virulence factors such as protease and bacterial cell surface proteins via a type IX secretion system (T9SS) that are involved in pathogenicity. P. melaninogenica also possesses all known orthologs of T9SS. In this study, a P. melaninogenica GAI 07411 mutant deficient in the orthologue of the T9SS‐encoding gene, porK, was constructed. Hemagglutination and biofilm formation were decreased in the porK mutant. Furthermore, following growth on skim milk‐containing medium, the diameters of the halos surrounding the porK mutant were smaller than those of the wild‐type strain, suggesting a decrease in secretion of proteases outside the bacterium. To investigate this in detail, culture supernatants of wild‐type and porK mutant strains were purified and compared by two‐dimensional electrophoresis. In the mutant strain, fewer spots were detected, indicating fewer secreted proteins. In infection experiments, the mortality rate of mice inoculated with the porK mutant strain was significantly lower than in the wild‐type strain. These results suggest that P. melaninogenica secretes potent virulence factors via the T9SS that contribute to its pathogenic ability.

     

Proteomannans in biofilm of Helicobacter pylori ATCC 43504

Background: The human bacterial pathogen Helicobacter pylori forms biofilms. However, the constituents of the biofilm have not been extensively investigated. In this study, we analyzed the carbohydrate and protein components of biofilm formed by H. pylori strain ATCC 43504 (NCTC 11637). Materials and Methods: Development of H. pylori biofilm was analyzed using scanning electron microscopy (SEM) and quantified using crystal violet staining. The extracted extracellular polysaccharide (EPS) matrix was analyzed using GC‐MS and nuclear magnetic resonance (NMR) analyses. Proteomic profiles of biofilms were examined by SDS–PAGE while deletion mutants of upregulated biofilm proteins were constructed and characterized. Results: Formation of H. pylori biofilm is time dependent as shown by crystal violet staining assay and SEM. NMR reveals the prevalence of 1,4‐mannosyl linkages in both developing and mature biofilms. Proteomic analysis of the biofilm indicates the upregulation of neutrophil‐activating protein A (NapA) and several stress‐induced proteins. Interestingly, the isogenic mutant napA revealed a different biofilm phenotype that showed reduced aggregated colonial structure when compared to the wild type. Conclusions: This in vitro study shows that mannose‐related proteoglycans (proteomannans) are involved in the process of H. pylori biofilm formation while the presence of upregulated NapA in the biofilm implies the potency to increase adhesiveness of H. pylori biofilm. Being a complex matrix of proteins and carbohydrates, which are probably interdependent, the H. pylori biofilm could possibly offer a protective haven for the survival of this gastric bacterial pathogen in the extragastric environments.     

HrpM is involved in glucan biosynthesis,biofilm formation and pathogenicity in Xanthomonas citri ssp. citri

Xanthomonas citri ssp. citri (Xcc) is the causal agent of citrus canker. This bacterium develops a characteristic biofilm on both biotic and abiotic surfaces. A biofilm‐deficient mutant was identified in a screening of a transposon mutagenesis library of the Xcc 306 strain constructed using the commercial Tn5 transposon EZ‐Tn5 <KAN‐2> Tnp Transposome (Epicentre). Sequence analysis of a mutant obtained in the screening revealed that a single copy of the EZ‐Tn5 was inserted at position 446 of hrpM, a gene encoding a putative enzyme involved in glucan synthesis. We demonstrate for the first time that the product encoded by the hrpM gene is involved in β‐1,2‐glucan synthesis in Xcc. A mutation in hrpM resulted in no disease symptoms after 4 weeks of inoculation in lemon and grapefruit plants. The mutant also showed reduced ability to swim in soft agar and decreased resistance to H ₂ O ₂ in comparison with the wild‐type strain. All defective phenotypes were restored to wild‐type levels by complementation with the plasmid pBBR1‐MCS containing an intact copy of the hrpM gene and its promoter. These results indicate that the hrpM gene contributes to Xcc growth and adaptation in its host plant.