Broad-host-range shuttle vectors for screening of regulated promoter activity in viridans group streptococci: isolation of a pH-regulated promoter

Viridans group streptococci are major constituents of the normal human oral flora and are also identified as the predominant pathogenic bacteria in native valve infective endocarditis. Little information is available regarding the regulation of gene expression in viridans group streptococci, either in response to changes in the oral environment or during development of endocarditis. We therefore constructed a set of broad-host-range vectors for the isolation of promoters from viridans group streptococci that are activated by specific environmental stimuli in vitro or in vivo. A genomic library of Streptococcus gordonii strain CH1 was constructed in one of the new vectors, and this library was introduced into a homologous bacterium by using an optimized electroporation protocol for viridans group streptococci. Because viridans group streptococci entering the bloodstream from the oral cavity encounter an increase in pH, we selected promoters upregulated by this specific stimulus. One of the selected promoter sequences showed homology to the promoter region of the hydA gene from Clostridium acetobutylicum, the expression of which is known to be regulated by the environmental pH. The isolation of this pH-regulated promoter shows that S. gordonii can sense an increase in the environmental pH, which serves as a signal for bacterial gene activation. Furthermore, this demonstrates the usefulness of these new selection vectors in research on adaptive gene expression of viridans group streptococci and possibly also of other gram-positive bacteria.     

Expression of Green Fluorescent Protein in Streptococcus gordonii DL1 and Its Use as a Species-Specific Marker in Coadhesion with Streptococcus oralis 34 in Saliva-Conditioned Biofilms In Vitro

Streptococcus gordonii is one of the predominant streptococci in the biofilm ecology of the oral cavity. It interacts with other bacteria through receptor-adhesin complexes formed between cognate molecules on the surfaces of the partner cells. To study the spatial organization of S. gordonii DL1 in oral biofilms, we used green fluorescent protein (GFP) as a species-specific marker to identify S. gordonii in a two-species in vitro oral biofilm flowcell system. To drive expression of gfp, we isolated and characterized an endogenous S. gordonii promoter, PhppA, which is situated upstream of the chromosomal hppA gene encoding an oligopeptide-binding lipoprotein. A chromosomal chloramphenicol acetyltransferase (cat) gene fusion with PhppA was constructed and used to demonstrate that PhppA was highly active throughout the growth of bacteria in batch culture. A promoterless 0.8-kb gfp (′gfp) cassette was PCR amplified from pBJ169 and subcloned to replace the cat cassette downstream of the S. gordonii-derived PhppA in pMH109-HPP, generating pMA1. Subsequently, the PhppA-′gfp cassette was PCR amplified from pMA1 and subcloned into pDL277 and pVA838 to generate the Escherichia coli-S. gordonii shuttle vectors pMA2 and pMA3, respectively. Each vector was transformed into S. gordonii DL1 aerobically to ensure GFP expression. Flow cytometric analyses of aerobically grown transformant cultures were performed over a 24-h period, and results showed that GFP could be successfully expressed in S. gordonii DL1 from PhppA and that S. gordonii DL1 transformed with the PhppA-′gfp fusion plasmid stably maintained the fluorescent phenotype. Fluorescent S. gordonii DL1 transformants were used to elucidate the spatial arrangement of S. gordonii DL1 alone in biofilms or with the coadhesion partner Streptococcus oralis 34 in two-species biofilms in a saliva-conditioned in vitro flowcell system. These results show for the first time that GFP expression in oral streptococci can be used as a species-specific marker in model oral biofilms.     

Oral Streptococci Utilize a Siglec-Like Domain of Serine-Rich Repeat Adhesins to Preferentially Target Platelet Sialoglycans in Human Blood

Damaged cardiac valves attract blood-borne bacteria, and infective endocarditis is often caused by viridans group streptococci. While such bacteria use multiple adhesins to maintain their normal oral commensal state, recognition of platelet sialoglycans provides an intermediary for binding to damaged valvular endocardium. We use a customized sialoglycan microarray to explore the varied binding properties of phylogenetically related serine-rich repeat adhesins, the GspB, Hsa, and SrpA homologs from Streptococcus gordonii and Streptococcus sanguinis species, which belong to a highly conserved family of glycoproteins that contribute to virulence for a broad range of Gram-positive pathogens. Binding profiles of recombinant soluble homologs containing novel sialic acid-recognizing Siglec-like domains correlate well with binding of corresponding whole bacteria to arrays. These bacteria show multiple modes of glycan, protein, or divalent cation-dependent binding to synthetic glycoconjugates and isolated glycoproteins in vitro. However, endogenous asialoglycan-recognizing clearance receptors are known to ensure that only fully sialylated glycans dominate in the endovascular system, wherein we find these particular streptococci become primarily dependent on their Siglec-like adhesins for glycan-mediated recognition events. Remarkably, despite an excess of alternate sialoglycan ligands in cellular and soluble blood components, these adhesins selectively target intact bacteria to sialylated ligands on platelets, within human whole blood. These preferred interactions are inhibited by corresponding recombinant soluble adhesins, which also preferentially recognize platelets. Our data indicate that circulating platelets may act as inadvertent Trojan horse carriers of oral streptococci to the site of damaged endocardium, and provide an explanation why it is that among innumerable microbes that gain occasional access to the bloodstream, certain viridans group streptococci have a selective advantage in colonizing damaged cardiac valves and cause infective endocarditis.     

Plant tissue-specific promoters can drive gene expression in Escherichia coli

Plant transgenesis often requires the use of tissue-specific promoters to drive the transgene expression exclusively in targeted tissues. Although the eukaryotic promoters are expected to stay silent in Escherichia coli, when the promoter-transgene units within the plant transformation vectors are constructed and propagated, some eukaryotic promoters have been reported to be active in prokaryotes. The potential activity of plant promoter in E. coli cells should be considered in cases of expression of proteins that are toxic for host cells, environmental risk assessment or the stability in E. coli of plant vectors for specific Cre/loxP applications. In this study, DNA fragments harbouring four embryo- and/or pollen-specific Arabidopsis thaliana promoters were investigated for their ability to drive heterologous gene expression in E. coli cells. For this, they were fused to gfp:gus reporter genes in the pCAMBIA1304 vector. Although BPROM, bacterial sigma70 promoter recognition program identified several sequences with characteristics similar to bacterial promoters including -10 and -35 sequences in each of tested fragments, the experimental approach showed that only one promoter fragment was able to drive relatively strong- and one promoter fragment relatively weak-GUS expression in E. coli cells. Remaining two tested promoters did not drive any transgene expression in bacteria. Our results also showed that cloning of a shorter plant promoter sequence into vectors containing lacZ α-complementation system can increase the probability of gene expression driven by upstream located lac promoter. This should be considered when cloning of plant expression units, the expression of which is unwanted in E. coli.     

Characterization of a Streptococcus sp.-Veillonella sp. community micromanipulated from dental plaque

Streptococci and veillonellae occur in mixed-species colonies during formation of early dental plaque. One factor hypothesized to be important in assembly of these initial communities is coaggregation (cell-cell recognition by genetically distinct bacteria). Intrageneric coaggregation of streptococci occurs when a lectin-like adhesin on one streptococcal species recognizes a receptor polysaccharide (RPS) on the partner species. Veillonellae also coaggregate with streptococci. These genera interact metabolically; lactic acid produced by streptococci is a carbon source for veillonellae. To transpose these interactions from undisturbed dental plaque to an experimentally tractable in vitro biofilm model, a community consisting of RPS-bearing streptococci juxtaposed with veillonellae was targeted by quantum dot-based immunofluorescence and then micromanipulated off the enamel surface and cultured. Besides the expected antibody-reactive cell types, a non-antibody-reactive streptococcus invisible during micromanipulation was obtained. The streptococci were identified as Streptococcus oralis (RPS bearing) and Streptococcus gordonii (adhesin bearing). The veillonellae could not be cultivated; however, a veillonella 16S rRNA gene sequence was amplified from the original isolation mixture, and this sequence was identical to the sequence of the previously studied organism Veillonella sp. strain PK1910, an oral isolate in our culture collection. S. oralis coaggregated with S. gordonii by an RPS-dependent mechanism, and both streptococci coaggregated with PK1910, which was used as a surrogate during in vitro community reconstruction. The streptococci and strain PK1910 formed interdigitated three-species clusters when grown as a biofilm using saliva as the nutritional source. PK1910 grew only when streptococci were present. This study confirms that RPS-mediated intrageneric coaggregation occurs in the earliest stages of plaque formation by bringing bacteria together to create a functional community.     

High Level Constitutive Expression of Luciferase Reporter by lsd90 Promoter in Fission Yeast

Because of a large number of molecular similarities with higher eukaryotes, the fission yeast Schizosaccharomyces pombe has been considered a potentially ideal host for expressing human proteins having therapeutic and pharmaceutical applications. However, efforts in this direction are hampered by lack of a strong promoter. Here, we report the isolation and characterization of a strong, constitutive promoter from S. pombe. A new expression vector was constructed by cloning the putative promoter region of the lsd90 gene (earlier reported to be strongly induced by heat stress) into a previously reported high copy number vector pJH5, which contained an ARS element corresponding to the mat2P flanking region and a truncated URA3m selectable marker. The resulting vector was used to study and compare the level of expression of the luciferase reporter with that achieved with the known vectors containing regulatable promoter nmt1 and the strong constitutive promoter adh1 in S. pombe and the methanol-inducible AOX1 promoter in Pichia pastoris. Following growth in standard media the new vector containing the putative lsd90 promoter provided constitutive expression of luciferase, at a level, which was 19-, 39- and 10-fold higher than that achieved with nmt1, adh1 and AOX1 promoters, respectively. These results indicate a great potential of the new lsd90 promoter-based vector for commercial scale expression of therapeutic proteins in S. pombe.     

Environment and Colonisation Sequence Are Key Parameters Driving Cooperation and Competition between Pseudomonas aeruginosa Cystic Fibrosis Strains and Oral Commensal Streptococci

Cystic fibrosis (CF) patient airways harbour diverse microbial consortia that, in addition to the recognized principal pathogen Pseudomonas aeruginosa, include other bacteria commonly regarded as commensals. The latter include the oral (viridans) streptococci, which recent evidence indicates play an active role during infection of this environmentally diverse niche. As the interactions between inhabitants of the CF airway can potentially alter disease progression, it is important to identify key cooperators/competitors and environmental influences if therapeutic intervention is to be improved and pulmonary decline arrested. Importantly, we recently showed that virulence of the P. aeruginosa Liverpool Epidemic Strain (LES) could be potentiated by the Anginosus-group of streptococci (AGS). In the present study we explored the relationships between other viridans streptococci (Streptococcus oralis, Streptococcus mitis, Streptococcus gordonii and Streptococcus sanguinis) and the LES and observed that co-culture outcome was dependent upon inoculation sequence and environment. All four streptococcal species were shown to potentiate LES virulence factor production in co-culture biofilms. However, in the case of S. oralis interactions were environmentally determined; in air cooperation within a high cell density co-culture biofilm occurred together with stimulation of LES virulence factor production, while in an atmosphere containing added CO₂ this species became a competitor antagonising LES growth through hydrogen peroxide (H₂O₂) production, significantly altering biofilm population dynamics and appearance. Streptococcus mitis, S. gordonii and S. sanguinis were also capable of H2O2 mediated inhibition of P. aeruginosa growth, but this was only visible when inoculated as a primary coloniser prior to introduction of the LES. Therefore, these observations, which are made in conditions relevant to the biology of CF disease pathogenesis, show that the pathogenic and colonisation potential of P. aeruginosa isolates can be modulated positively and negatively by the presence of oral commensal streptococci.     

Synthetic promoter libraries for Corynebacterium glutamicum

The ability to modulate gene expression is an important genetic tool in systems biology and biotechnology. Here, we demonstrate that a previously published easy and fast PCR-based method for modulating gene expression in lactic acid bacteria is also applicable to Corynebacterium glutamicum. We constructed constitutive promoter libraries based on various combinations of a previously reported C. glutamicum -10 consensus sequence (gngnTA(c/t)aaTgg) and the Escherichia coli -35 consensus, either with or without an AT-rich region upstream. A promoter library based on consensus sequences frequently found in low-GC Gram-positive microorganisms was also included. The strongest promoters were found in the library with a -35 region and a C. glutamicum -10 consensus, and this library also represents the largest activity span. Using the alternative -10 consensus TATAAT, which can be found in many other prokaryotes, resulted in a weaker but still useful promoter library. The upstream AT-rich region did not appear to affect promoter strength in C. glutamicum. In addition to the constitutive promoters, a synthetic inducible promoter library, based on the E. coli lac-promoter, was constructed by randomizing the 17-bp spacer between -35 and -10 consensus sequences and the sequences surrounding these. The inducible promoter library was shown to result in β-galactosidase activities ranging from 284 to 1,665 Miller units when induced by IPTG, and the induction fold ranged from 7–59. We find that the synthetic promoter library (SPL) technology is convenient for modulating gene expression in C. glutamicum and should have many future applications, within basic research as well as for optimizing industrial production organisms.     

Use of the Lactococcal nisA Promoter To Regulate Gene Expression in Gram-Positive Bacteria: Comparison of Induction Level and Promoter Strength

We characterized the regulated activity of the lactococcal nisA promoter in strains of the gram-positive species Streptococcus pyogenes, Streptococcus agalactiae, Streptococcus pneumoniae, Enterococcus faecalis, and Bacillus subtilis. nisA promoter activity was dependent on the proteins NisR and NisK, which constitute a two-component signal transduction system that responds to the extracellular inducer nisin. The nisin sensitivity and inducer concentration required for maximal induction varied among the strains. Significant induction of the nisA promoter (10- to 60-fold induction) was obtained in all of the species studied at a nisin concentration just below the concentration at which growth is inhibited. The efficiency of the nisA promoter was compared to the efficiencies of the Spac, xylA, and lacA promoters in B. subtilis and in S. pyogenes. Because nisA promoter-driven expression is regulated in many gram-positive bacteria, we expect it to be useful for genetic studies, especially studies with pathogenic streptococci in which no other regulated promoters have been described.     

Novel plasmid-based genetic tools for the study of promoters and terminators in Streptococcus pneumoniae and Enterococcus faecalis

Promoter-probe and terminator-probe plasmid vectors make possible to rapidly examine whether particular sequences function as promoter or terminator signals in various genetic backgrounds and under diverse environmental stimuli. At present, such plasmid-based genetic tools are very scarce in the Gram-positive pathogenic bacteria Streptococcus pneumoniae and Enterococcus faecalis. Hence, we developed novel promoter-probe and terminator-probe vectors based on the Streptococcus agalactiae pMV158 plasmid, which replicates autonomously in numerous Gram-positive bacteria. As reporter gene, a gfp allele encoding a variant of the green fluorescent protein was used. These genetic tools were shown to be suitable to assess the activity of promoters and terminators (both homologous and heterologous) in S. pneumoniae and E. faecalis. In addition, the promoter-probe vector was shown to be a valuable tool for the analysis of regulated promoters in vivo, such as the promoter of the pneumococcal fuculose kinase gene. These new plasmid vectors will be very useful for the experimental verification of predicted promoter and terminator sequences, as well as for the construction of new inducible-expression vectors. Given the promiscuity exhibited by the pMV158 replicon, these vectors could be used in a variety of Gram-positive bacteria.     

A Comparative Analysis of Constitutive Promoters Located in Adeno-Associated Viral Vectors

The properties of constitutive promoters within adeno-associated viral (AAV) vectors have not yet been fully characterized. In this study, AAV vectors, in which enhanced GFP expression was directed by one of the six constitutive promoters (human β-actin, human elongation factor-1α, chicken β-actin combined with cytomegalovirus early enhancer, cytomegalovirus (CMV), simian virus 40, and herpes simplex virus thymidine kinase), were constructed and introduced into the HCT116, DLD-1, HT-1080, and MCF-10A cell lines. Quantification of GFP signals in infected cells demonstrated that the CMV promoter produced the highest GFP expression in the six promoters and maintained relatively high GFP expression for up to eight weeks after infection of HCT116, DLD-1, and HT-1080. Exogenous human CDKN2A gene expression was also introduced into DLD-1 and MCF-10A in a similar pattern by using AAV vectors bearing the human β-actin and the CMV promoters. The six constitutive promoters were subsequently placed upstream of the neomycin resistance gene within AAV vectors, and HCT116, DLD-1, and HT-1080 were infected with the resulting vectors. Of the six promoters, the CMV promoter produced the largest number of G418-resistant colonies in all three cell lines. Because AAV vectors have been frequently used as a platform to construct targeting vectors that permit gene editing in human cell lines, we lastly infected the three cell lines with AAV-based targeting vectors against the human PIGA gene in which one of the six promoters regulate the neomycin resistance gene. This assay revealed that the CMV promoter led to the lowest PIGA gene targeting efficiency in the investigated promoters. These results provide a clue to the identification of constitutive promoters suitable to express exogenous genes with AAV vectors, as well as those helpful to conduct efficient gene targeting using AAV-based targeting vectors in human cell lines.     

Promoter cassettes, antibiotic-resistance genes, and vectors for plant transformation

We have constructed a set of plant transformation vectors, promoter cassettes, and chimeric antibiotic-resistance genes for the transformation and expression of foreign genes in plants sensitive to Agrobacterium infection. The different vectors allow for either concurrent or consecutive selection for kanamycin and hygromycin resistance and have a number of unique restriction sites for the insertion of additional DNA. The promoter cassettes utilize the CaMV 19S and CaMV 35S promoters and are constructed to allow for the easy insertion of foreign genes. The cloned gene can then easily be inserted into the transformation vectors. We have utilized the promoter cassettes to express the hygromycin-resistance gene either from the CaMV 35S or the CaMV 19S promoters, with both chimeric resistance genes allowing for the selection of hygromycin-resistant tobacco plants.     

Interactions between Oral Bacteria: Inhibition of Streptococcus mutans Bacteriocin Production by Streptococcus gordonii

Streptococcus mutans has been recognized as an important etiological agent in human dental caries. Some strains of S. mutans also produce bacteriocins. In this study, we sought to demonstrate that bacteriocin production by S. mutans strains GS5 and BM71 was mediated by quorum sensing, which is dependent on a competence-stimulating peptide (CSP) signaling system encoded by the com genes. We also demonstrated that interactions with some other oral streptococci interfered with S. mutans bacteriocin production both in broth and in biofilms. The inhibition of S. mutans bacteriocin production by oral bacteria was stronger in biofilms than in broth. Using transposon Tn916 mutagenesis, we identified a gene (sgc; named for Streptococcus gordonii challisin) responsible for the inhibition of S. mutans bacteriocin production by S. gordonii Challis. Interruption of the sgc gene in S. gordonii Challis resulted in attenuated inhibition of S. mutans bacteriocin production. The supernatant fluids from the sgc mutant did not inactivate the exogenous S. mutans CSP as did those from the parent strain Challis. S. gordonii Challis did not inactivate bacteriocin produced by S. mutans GS5. Because S. mutans uses quorum sensing to regulate virulence, strategies designed to interfere with these signaling systems may have broad applicability for biological control of this caries-causing organism.     

Application of the resident plasmid integration technique to construct a strain of Streptococcus godronii able to express the Bacillus circulans cycloisomaltooligosaccharide glucanotransferase gene,and secrete its active gene product

A novel transformation technique, resident plasmid integration, for the cloning of foreign DNA in oral streptococci was described recently (T. Shiroza and H. K. Kuramitsu, Plasmid, 1995, 34, 85–95). This technique is based on the integration of linearized foreign genes by recombination-proficient bacteria onto a resident plasmid, if an appropriate selection marker is flanked by the same anchor sites present in the resident plasmid. Since the transforming vehicles for this system included a pUC-derived replication origin, the high level expression in Escherichia coli cells hindered the cloning of certain genes. In the present study, new plasmids were constructed, two resident plasmids, four integration plasmids, and four cloning plasmids, all of which possess the medium-copy number replication origin, p15A ori, isolated from pACYC177. The resident plasmids consisted of the following three components: the p15A ori (0.65-kb BglII fragment), the pVA380-1 basic replicon functional in mutans streptococci (2.5-kb BamHI fragment), and either an erythromycin resistance or a spectinomycin resistance gene (0.9- or 1.1-kb BamHI fragment, respectively). Most of the basic replicon of pVA380-1, except for the 3′-portion of the 0.2-kb region, in the resident plasmid was replaced with a kanamycin resistance gene to construct the four integration plasmids. Therefore, the upstream and downstream anchor sites for the double cross-over event in this new system were 0.65-kb p15A ori and the 0.2-kb portion of the 3′-end of pVA380-1 replicon, respectively. This system was used to clone the gene coding for cycloisomaltooligosaccharide glucanotransferase which produces cycloisomaltooligosaccharide, a potent inhibitor of oral streptococcal glucosyltransferase, isolated from Bacillus circulans chromosome, into Streptococcus gordonii, and its gene product was successfully secreted into the culture media. Plasmids described here should be useful tools for introducing heterologous DNA into resident plasmids following integration in oral streptococci.     

Functional Analysis and Randomization of the Nisin-Inducible Promoter for Tuning Gene Expression in Lactococcus lactis

Three putative promoter regions were identified preceding the nisZ gene in Lactococcus lactis HSM-22. To investigate their function in the control of nisZ biosynthesis, green fluorescence protein (GFP) was adopted as probe to determine activities of the three promoters. The results showed that PnisZ-0 containing two sets of the ?35 and ?10 regions exhibited the same maximum activity as promoter PnisZ-2 containing the putative promoter region near the start codon. However, the GFP expression level directed by PnisZ-0 was twofold higher than that found with PnisZ-2 under low-dose nisin, indicating that promoter PnisZ-1 distant from the start codon could be important in response to the inducer nisin. Then, Pnis-2 was randomized to develop functional promoters through the degenerate oligonucleotide approach in L. lactis. 35 inducible promoters and 14 constitutive promoters were obtained, covering 3–5 logs of expression levels in small increments of activity. Sequence analysis revealed that base changes in both consensus sequence and spacing sequence resulted in remarkable decrease of promoter activity, while the sequence outside ?35 and ?10 regions would influence the promoter function radically. The functional promoters were evaluated for the efficiency and stability to control β-galactosidase (Gal) expression in L. lactis. High correlation was obtained between the Gal activity and promoter strength, suggesting that promoters developed here have the potential for fine tuning gene expression in L. lactis.     

The Interrelationship between Promoter Strength,Gene Expression,and Growth Rate

In exponentially growing bacteria, expression of heterologous protein impedes cellular growth rates. Quantitative understanding of the relationship between expression and growth rate will advance our ability to forward engineer bacteria, important for metabolic engineering and synthetic biology applications. Recently, a work described a scaling model based on optimal allocation of ribosomes for protein translation. This model quantitatively predicts a linear relationship between microbial growth rate and heterologous protein expression with no free parameters. With the aim of validating this model, we have rigorously quantified the fitness cost of gene expression by using a library of synthetic constitutive promoters to drive expression of two separate proteins (eGFP and amiE) in E. coli in different strains and growth media. In all cases, we demonstrate that the fitness cost is consistent with the previous findings. We expand upon the previous theory by introducing a simple promoter activity model to quantitatively predict how basal promoter strength relates to growth rate and protein expression. We then estimate the amount of protein expression needed to support high flux through a heterologous metabolic pathway and predict the sizable fitness cost associated with enzyme production. This work has broad implications across applied biological sciences because it allows for prediction of the interplay between promoter strength, protein expression, and the resulting cost to microbial growth rates.     

低温菌启动子探针质粒的构建

为了在宿主菌Acinetobacter sp.DWC6中构建低温菌蛋白表达载体,以pBR322质粒为基础,去除质粒上β-内酰胺酶基因的启动子片段,取而代之为来源于质粒pJRD215的卡那霉素抗性基因片段,并在pBR322中插入Acinetobacter菌属特异性ori的DNA片段,构建了能在Acinetobacter sp.DWC6和E.coli中正常复制的启动子探针质粒pBAP1。通过在质粒pBAP1中的β-内酰胺酶基因上游随机导入Acinetobacter sp.DWC6基因组片段,通过检测宿主细胞的氨苄青霉素抗性和β-内酰胺酶活性,来筛选强启动子片段,并分析了启动子探针质粒载体的功能及启动子的强度。