The Sialic Acid Binding Protein,Hsa, in Streptococcus gordonii DL1 also Mediates Intergeneric Coaggregation with Veillonella Species

Dental biofilm development involves initial colonization of the tooth’s surface by pioneer colonizers, followed by cell-cell coaggregation between the pioneer and later colonizers. Streptococcus gordonii is one of the pioneer colonizers. In addition to its role in oral biofilm development, S. gordonii also is a pathogen in infective endocarditis in susceptible humans. A surface adhesin, Hsa, has been shown to play a critical role in colonization of S. gordonii on the heart tissue; however, its role in oral biofilm development has not been reported. In this study we demonstrate that Hsa is essential for coaggregation between S. gordonii and Veillonella sp., which are bridging species connecting the pioneer colonizers to the late colonizers. Interestingly, the same domains shown to be required for Hsa binding to sialic acid on the human cell surface are also required for coaggregation with Veillonella sp. However, sialic acid appeared not to be required for this intergeneric coaggregation. This result suggests that although the same domains of Hsa are involved in binding to eukaryotic as well as Veillonella cells, the binding mechanism is different. The gene expression pattern of hsa was also studied and shown not to be induced by coaggregation with Veillonella sp.     

嗜盐四联球菌arc operon多拷贝基因在精氨酸代谢中的作用

【背景】嗜盐四联球菌(Tetragenococcus halophilus)是一类存在于发酵食品中的耐盐乳酸菌,研究其精氨酸(arginine,Arg)代谢对解析食品发酵过程中氨基甲酸乙酯(ethyl carbamate,EC)前体积累机制和保障食品安全具有重要意义。【目的】研究酱醪来源嗜盐四联球菌精氨酸脱亚氨基(arginine deiminase,ADI)途径的基因构成,揭示这些基因对菌株精氨酸代谢和氨基甲酸乙酯前体瓜氨酸(citrulline,Cit)利用与积累的影响。【方法】采用PCR扩增与测序分析不同菌株的ADI途径基因组成,通过比较ADI途径关键基因转录水平和关键酶活性,探究环境因素对嗜盐四联球菌代谢氨基酸能力的影响及各拷贝基因参与氨基酸代谢的功能。【结果】酱醪来源嗜盐四联球菌基因组中ADI途径基因类型主要有两大类：以菌株R23为代表含有完整arc操纵子(operon)基因且具有最多基因拷贝数;以菌株C3为代表缺失arcA和arcB但含有多拷贝arcB和arcC。基因组中有arcA的菌株才具有利用精氨酸能力,并通过利用精氨酸生成瓜氨酸。体系中精氨酸含量和乙醇与脂肪酸的存在均可影响嗜盐四联球菌利用精氨酸积累中间产物瓜氨酸。当精氨酸含量大于5 g/L或体系中含有乙醇与脂肪酸时,嗜盐四联球菌会利用精氨酸积累中间产物瓜氨酸。脂肪酸和乙醇对ADI途径的3个关键酶均有显著抑制作用,可使精氨酸脱亚氨基酶(arginine deiminase,ADI)、鸟氨酸氨甲酰基转移酶(ornithine transcarbamylase,OTC)和氨甲酰磷酸激酶(carbamate kinase,CK)的活性分别降低41.0%、46.4%和60.0%。嗜盐四联球菌中arcB转录水平分别是其拷贝arcB₁和arcB₂的10.5倍和29.8倍,arcC的转录水平分别是arcC₁、arcC₂、arcC₃的17.6、20.3、23.9倍,说明arcB和arcC在瓜氨酸代谢中起主要作用。【结论】精氨酸含量和乙醇加脂肪酸是影响嗜盐四联球菌代谢精氨酸能否积累瓜氨酸的关键环境因素。嗜盐四联球菌arc operon的多拷贝基因中,arcB和arcC基因在瓜氨酸代谢中起主要作用。     

The nucleoid‐associated protein Fis directly modulates the synthesis of cellulose,an essential component of pellicle–biofilms in the phytopathogenic bacterium Dickeya dadantii

Bacteria use biofilm structures to colonize surfaces and to survive in hostile conditions, and numerous bacteria produce cellulose as a biofilm matrix polymer. Hence, expression of the bcs operon, responsible for cellulose biosynthesis, must be finely regulated in order to allow bacteria to adopt the proper surface‐associated behaviours. Here we show that in the phytopathogenic bacterium, Dickeya dadantii, production of cellulose is required for pellicle–biofilm formation and resistance to chlorine treatments. Expression of the bcs operon is growth phase‐regulated and is stimulated in biofilms. Furthermore, we unexpectedly found that the nucleoid‐associated protein and global regulator of virulence functions, Fis, directly represses bcs operon expression by interacting with an operator that is absent from the bcs operon of animal pathogenic bacteria and the plant pathogenic bacterium Pectobacterium. Moreover, production of cellulose enhances plant surface colonization by D. dadantii. Overall, these data suggest that cellulose production and biofilm formation may be important factors for surface colonization by D. dadantii and its subsequent survival in hostile environments. This report also presents a new example of how bacteria can modulate the action of a global regulator to co‐ordinate basic metabolism, virulence and modifications of lifestyle.     

Let there be bioluminescence: development of a biophotonic imaging platform for in situ analyses of oral biofilms in animal models

In the current study, we describe a novel biophotonic imaging‐based reporter system that is particularly useful for the study of virulence in polymicrobial infections and interspecies interactions within animal models. A suite of luciferase enzymes was compared using three early colonizing species of the human oral flora (Streptococcus mutans, Streptococcus gordonii and Streptococcus sanguinis) to determine the utility of the different reporters for multiplexed imaging studies in vivo. Using the multiplex approach, we were able to track individual species within a dual‐species oral infection model in mice with both temporal and spatial resolution. We also demonstrate how biophotonic imaging of multiplexed luciferase reporters could be adapted for real‐time quantification of bacterial gene expression in situ. By creating an inducible dual‐luciferase expressing reporter strain of S. mutans, we were able to exogenously control and measure expression of nlmAB (encoding the bacteriocin mutacin IV) within mice to assess its importance for the persistence ability of S. mutans in the oral cavity. The imaging system described in the current study circumvents many of the inherent limitations of current animal model systems, which should now make it feasible to test hypotheses that were previously impractical to model.     

Proteome Analysis Identifies the Dpr Protein of Streptococcus mutans as an Important Factor in the Presence of Early Streptococcal Colonizers of Tooth Surfaces

Oral streptococci are primary colonizers of tooth surfaces and Streptococcus mutans is the principal causative agent of dental caries in humans. A number of proteins are involved in the formation of monospecies biofilms by S. mutans. This study analyzed the protein expression profiles of S. mutans biofilms formed in the presence or absence of S. gordonii, a pioneer colonizer of the tooth surface, by two-dimensional gel electrophoresis (2-DE). After identifying S. mutans proteins by Mass spectrometric analysis, their expression in the presence of S. gordonii was analyzed. S. mutans was inoculated with or without S. gordonii DL1. The two species were compartmentalized using 0.2-μl Anopore membranes. The biofilms on polystyrene plates were harvested, and the solubilized proteins were separated by 2-DE. When S. mutans biofilms were formed in the presence of S. gordonii, the peroxide resistance protein Dpr of the former showed 4.3-fold increased expression compared to biofilms that developed in the absence of the pioneer colonizer. In addition, we performed a competition assay using S. mutans antioxidant protein mutants together with S. gordonii and other initial colonizers. Growth of the dpr-knockout S. mutans mutant was significantly inhibited by S. gordonii, as well as by S. sanguinis. Furthermore, a cell viability assay revealed that the viability of the dpr-defective mutant was significantly attenuated compared to the wild-type strain when co-cultured with S. gordonii. Therefore, these results suggest that Dpr might be one of the essential proteins for S. mutans survival on teeth in the presence of early colonizing oral streptococci.     

Proteomic investigation of the impact of oxygen on the protein profiles of hyaluronic acid‐producing Streptococcus zooepidemicus

Hyaluronic acid (HA) is a linear and negatively charged polysaccharide regularly used in medicine and cosmetics. Recently Streptococcus zooepidemicus has been exploited in the fermentation industry to produce HA. Many studies showed that higher amounts of HA were produced under aerobic condition compared to anaerobic conditions. To explore the effect of oxygen on the HA synthesis in S. zooepidemicus, 2‐DE was used to compare the proteomes of aerobically and anaerobically fermented bacteria to identify proteins, which might be associated with the influence of oxygen on the HA synthesis. Totally nine pairs of 2‐DE gels collected from three batches were compared and nine overexpressed proteins were observed in aerobically fermented bacteria. These proteins were identified by LC/tandem MS as dihydrolipoamide dehydrogenase, UDP‐acetyl‐glucosamine pyrophosphoylase, dihydrolipoamide‐S‐acetyltransferase and acetoin dehydrogenase α and β chains, respectively. These upregulated proteins were involved in acetoin dissimilation, the central carbon metabolism and the HA anabolic pathway, implicating that oxygen might augment the expression of genes that are involved in central energy metabolism, acetoin reutilization and HA biosynthesis to enhance the amount of acetyl‐CoA as such that more acetyl‐CoA can be diverged from the central carbon metabolism to replenish acetyl‐CoA for the HA synthesis.     

Interaction between Streptococcus spp. and Veillonella tobetsuensis in the Early Stages of Oral Biofilm Formation

Dental plaque is a multispecies oral biofilm, the development of which is initiated by adherence of the pioneer Streptococcus spp. Oral Veillonella spp., including V. atypica, V. denticariosi, V. dispar, V. parvula, V. rogosae, and V. tobetsuensis, are known as early colonizers in oral biofilm formation. These species have been reported to coaggregate with Streptococcus spp. in a metabolic cooperation-dependent manner to form biofilms in human oral cavities, especially in the early stages of biofilm formation. However, in our previous study, Streptococcus gordonii showed biofilm formation to the greatest extent in the presence of V. tobetsuensis, without coaggregation between species. These results suggest that V. tobetsuensis produces signaling molecules that promote the proliferation of S. gordonii in biofilm formation. It is well known in many bacterial species that the quorum-sensing (QS) system regulates diverse functions such as biofilm formation. However, little is known about the QS system with autoinducers (AIs) with respect to Veillonella and Streptococcus spp. Recently, autoinducer 1 (AI-1) and AI-2 were detected and identified in the culture supernatants of V. tobetsuensis as strong signaling molecules in biofilm formation with S. gordonii. In particular, the supernatant from V. tobetsuensis showed the highest AI-2 activity among 6 oral Veillonella species, indicating that AIs, mainly AI-2, produced by V. tobetsuensis may be important factors and may facilitate biofilm formation of S. gordonii. Clarifying the mechanism that underlies the QS system between S. gordonii and V. tobetsuensis may lead to the development of novel methods for the prevention of oral infectious diseases caused by oral biofilms.     

Tandem genes encode cell-surface polypeptides SspA and SspB which mediate adhesion of the oral bacterium Streptococcus gordonii to human and bacterial receptors

The highly conserved antigen I/II family of polypeptides produced by oral streptococci are believed to be colonization determinants and may mediate adhesion of bacterial cells to salivary glycoproteins adsorbed to cells and tissues in the human oral cavity. Streptococcus gordonii is shown to express, on the cell surface, two antigen I/II polypeptides designated SspA and SspB (formerly Ssp-5) that are the products of tandemly arranged chromosomal genes. The structure and arrangement of these genes is similar in two independently isolated strains, DL1 and M5, of S. gordonii. The mature polypeptide sequences of M5 SspA (1539 amino acid (aa) residues) and SspB (1462 aa residues) are almost wholly conserved (98% identical) in the C-terminal regions (from residues 796 in SspA and 719 in SspB, to the respective C-termini), well-conserved (84%) at the N-terminal regions (residues 1–429), and divergent (only 27% identical residues) within the intervening central regions. Insertional inactivation of the sspA gene in S. gordonii DL1 resulted in reduced binding of cells to salivary agglutinin glycoprotein (SAG), human erythrocytes, and to the oral bacterium Actinomyces naeslundii. Further reductions in streptococcal cell adhesion to SAG and to two strains of A. naeslundii were observed when both sspA and sspB genes were inactivated. The results suggest that both SspA and SspB polypeptides are involved in adhesion of S. gordonii cells to human and bacterial receptors.     

Isolation,identification and genetic organization of the ADI operon in <Emphasis Type="Italic">Enterococcus faecium</Emphasis> GR7

L-Arginine is an indispensable amino acid, as it is required for normal growth of microbes, plants and animals (Szende et al., Cancer Cell Int 1:1475–1480, 2001). Arginine deiminase is the first enzyme of arginine deiminase (ADI) pathway, which catalyzes the conversion of arginine to citrulline and ammonia in an irreversible reaction. Lactic acid bacteria isolated from dairy products were investigated for their ability to hydrolyze arginine. Citrulline production in many LAB strains suggests that the arginine metabolism takes place via the arginine deiminase pathway. The highest arginine deiminase specific activity (0.27 IU/mg) was reported in isolate GR7, which was characterized morphologically, biochemically and by 16S rRNA gene sequencing as Enterococcus faecium. Genetic organization of the ADI operon in E. faecium GR7 was further studied using various molecular biology and computational techniques. Sequence analysis revealed that the genes involved in arginine catabolism are clustered together in an operon (3,906 bp) consisting of the genes arcA (arginine deiminase), arcB (ornithine transcarbamylase), and arcC (carbamate kinase), which are localized on the anti-sense strand of genomic DNA. Nucleotide sequence analysis revealed three open reading frames (ORFs) that were arranged contiguously and transcribed in the same direction, as an apparent operon. The genes followed the order arcC, arcB, arcA, which differs from that found in other microorganisms. The information obtained in this study provides the basis for testing the potential of arginine catabolism to control the emergence of arginine auxotrophic tumors.     

Thermostability, oligomerization and DNA-binding properties of the regulatory protein ArgR from the hyperthermophilic bacterium Thermotoga neapolitana

The hexameric regulatory protein ArgR formed by arginine-mediated dimerization of identical trimers governs the expression of genes required for arginine metabolism and some other genes in mesophilic and moderately thermophilic bacteria. We have cloned the argR gene from two hyperthermophilic bacteria of the genus Thermotoga. The two-domain ArgR proteins encoded by T. neapolitana and T. maritima share a low degree of sequence similarity with other bacterial arginine repressors. The ArgR protein from T. neapolitana binds to an operator located just upstream of its coding sequence and, therefore, the argR gene may be autoregulated. The protein has extremely high intrinsic thermostability and tolerance to urea. Moreover, its binding to target DNA increases the melting temperature by approximately 15° C. The formation of oligomeric ArgR-DNA complexes is a function of protein concentration, with hexameric complexes being favoured at higher concentrations. In the presence of arginine the hyperthermophilic ArgR protein binds to its own operator, argRo, only by forming hexamer ArgR-DNA complexes, whereas both trimer-DNA and hexamer-DNA complexes are detected in the absence of arginine. However, the affinity of T. neapolitana ArgR for DNA has been found to be higher for a mixture of trimers and non-bound hexamers than for arginine-bound hexamers. Our data indicate that genes for arginine biosynthesis are clustered in a putative operon, which could also be regulated by the ArgR protein, in the hyperthermophilic host. Received: 19 July 1999 / Accepted: 4 November 1999