Gene Expression In Streptococcus Research Articles

The importance of pathogenicity factors of some Streptococcus spp. and Klebsiella spp. in determining their etiological role in the inflammatory processes of the respiratory tract

Together with the known pathogens of inflammatory processes of the respiratory tract in clinical practice are often found representatives of Streptococcus and Klebsiella, previously considered commensals of the mucous membranes of the upper respiratory tract. The exchange of genetic information facilitates the transfer of virulence factors between strains not only within the species but also within the genus. In such cases, the acquisition of virulence genes by nonpathogenic species from representatives of pathogenic species contributes to the manifestation of previously not typical properties. Therefore, the aim of the research was to study the virulence of opportunistic Streptococcus spp. and Klebsiella spp. in inflammatory processes of the respiratory tract and substantiate their etiological role in the development of the disease. We studied 220 strains of Streptococcus spp. and 97 strains of Klebsiella spp., isolated from patients with inflammatory processes in the respiratory tract and from healthy individuals. Strains of Streptococcus spp. were investigated for the presence of virulence genes: sagA, lmb, fapl, ply, lytA. Strains of Klebsiella spp. were examined for the presence of virulence genes: MrkD, magA, kfu. The phenotypic marker of lmb gene expression in Streptococcus and MrkD gene in Klebsiella was the indicator of adhesion of isolated strains to buccal epithelial cells. Expression of the fapl gene was evaluated in a phenotypic biofilm formation test. In individuals with upper respiratory tract inflammation, the most common types of strepto -cocci were: S. mitis, S. anginosus, and S. oralis. Strains of these species isolated from inflammatory processes in the upper respiratory tract had 2—4 times greater adhesiveness than strains isolated from healthy individuals. Phenotypic determination of the ability to biofilm formation showed that strains of Streptococcus containing the fapl gene formed a dense biofilm in contrast to strains without the fapl gene. K. oxytoca strains isolated from people with sinusitis had mrkd, magA, and kfu virulence genes that are characteristic of K. pneumoniae strains. In phenotypic tests, it was found that the value of the adhesion index in K. oxytoca strains isolated from patients is 4 times higher than in strains of this species isolated from healthy individuals. Thus, to confirm the etiological role of an opportunistic microorganism in the development of the infectious process, it is necessary to be guided by data on the genetic and phenotypic markers of virulence of the isolated strain.

NADH-Mediated Gene Expression in Streptococcus pneumoniae and Role of Rex as a Transcriptional Repressor of the Rex-Regulon

Nicotinamide adenine dinucleotides (NAD(H)) play a vital role in various biological processes, including keeping the cellular redox balance. In this study, we investigate the regulatory responses of Streptococcus pneumoniae D39 to NADH and characterize the role of the Rex protein as a transcriptional repressor of the gapN, fba, pncB, adhB2, gap, and adhE genes. Transcriptomic analysis was used to observe the response of S. pneumoniae D39 to NADH. Our microarray studies revealed elevated expression of various genes/operons involved in transport and biosynthesis of niacin (gapN, fba, pncB, adhB2, gap, and adhE). Promoter lacZ-fusion assays and microarray studies with the rex mutant revealed the role of Rex as a transcriptional repressor of gapN, fba, pncB, adhB2, gap, and adhE involved in niacin uptake and biosynthesis, in the presence of NADH. We predict the operator site (5′-TTGTKAWAAWWTTCACAA-3′) of Rex in the regulatory regions of Rex-regulated genes that was subsequently validated by promoter mutational experiments.

A tetracycline-inducible integrative expression system for Streptococcus pneumoniae.

Inducible gene expression systems are very useful to analyze cellular processes. The ability to switch the expression state of genes of interest may even be crucial if essential traits or genetic instability are involved. An integrative plasmid, pTEX2, was designed using the (anhydro)tetracycline-inducible promoter Pxyl/tet from staphylococcal plasmid pRAB11 to control gene expression in Streptococcus pneumoniae. The system was evaluated by expressing genes of the two-component regulatory system ciaRH of S. pneumoniae. With full induction of Pxyl/tet, wild-type levels of the response regulator CiaR were obtained, while the uninduced basal expression was low. Hyperactive variants of the kinase gene ciaH normally causing pronounced genetic instability could be handled without any problems upon cloning into pTEX2. Therefore, the expression system is well suited to express physiological levels of proteins in S. pneumoniae and also to aid regulatory studies.

Co(2+)-dependent gene expression in Streptococcus pneumoniae: opposite effect of Mn(2+) and Co(2+) on the expression of the virulence genes psaBCA, pcpA, and prtA.

Manganese (Mn2+)-, zinc (Zn2+)- and copper (Cu2+) play significant roles in transcriptional gene regulation, physiology, and virulence of Streptococcus pneumoniae. So far, the effect of the important transition metal ion cobalt (Co2+) on gene expression of S. pneumoniae has not yet been explored. Here, we study the impact of Co2+ stress on the transcriptome of S. pneumoniae strain D39. BLAST searches revealed that the genome of S. pneumoniae encodes a putative Co2+-transport operon (cbi operon), the expression of which we show here to be induced by a high Co2+ concentration. Furthermore, we found that Co2+, as has been shown previously for Zn2+, can cause derepression of the genes of the PsaR virulence regulon, encoding the Mn2+-uptake system PsaBCA, the choline binding protein PcpA and the cell-wall associated serine protease PrtA. Interestingly, although Mn2+ represses expression of the PsaR regulon and Co2+ leads to derepression, both metal ions stimulate interaction of PsaR with its target promoters. These data will be discussed in the light of previous studies on similar metal-responsive transcriptional regulators.

Sialic acid-mediated gene expression in Streptococcus pneumoniae and role of NanR as a transcriptional activator of the nan gene cluster.

In this study, we investigated the transcriptomic response of Streptococcus pneumoniae D39 to sialic acid (N-acetylneuraminic acid [Neu5Ac]). Transcriptome comparison of wild-type D39 grown in M17 medium with and without sialic acid revealed the elevated expression of various genes and operons, including the nan gene cluster (nan operon I and nanA gene). Our microarray analysis and promoter-lacZ fusion studies showed that the transcriptional regulator NanR acts as a transcriptional activator of nan operon I and the nanA gene in the presence of sialic acid. The putative regulatory site of NanR in the promoter region of nan operon I is predicted and confirmed by promoter truncation experiments. Furthermore, the role of CcpA in the regulation of the nan gene cluster is demonstrated through microarray analysis and promoter-lacZ fusion studies, suggesting that in the presence of sialic acid and glucose, CcpA represses the expression of nan operon I while the expression of the nanA gene is CcpA independent.

Control of transcription elongation by GreA determines rate of gene expression in Streptococcus pneumoniae.

Transcription by RNA polymerase may be interrupted by pauses caused by backtracking or misincorporation that can be resolved by the conserved bacterial Gre-factors. However, the consequences of such pausing in the living cell remain obscure. Here, we developed molecular biology and transcriptome sequencing tools in the human pathogen Streptococcus pneumoniae and provide evidence that transcription elongation is rate-limiting on highly expressed genes. Our results suggest that transcription elongation may be a highly regulated step of gene expression in S. pneumoniae. Regulation is accomplished via long-living elongation pauses and their resolution by elongation factor GreA. Interestingly, mathematical modeling indicates that long-living pauses cause queuing of RNA polymerases, which results in ‘transcription traffic jams’ on the gene and thus blocks its expression. Together, our results suggest that long-living pauses and RNA polymerase queues caused by them are a major problem on highly expressed genes and are detrimental for cell viability. The major and possibly sole function of GreA in S. pneumoniae is to prevent formation of backtracked elongation complexes.

Role of global and local topology in the regulation of gene expression in Streptococcus pneumoniae.

The most basic level of transcription regulation in Streptococcus pneumoniae is the organization of its chromosome in topological domains. In response to drugs that caused DNA-relaxation, a global transcriptional response was observed. Several chromosomal domains were identified based on the transcriptional response of their genes: up-regulated (U), down-regulated (D), non-regulated (N), and flanking (F). We show that these distinct domains have different expression and conservation characteristics. Microarray fluorescence units under non-relaxation conditions were used as a measure of gene transcriptional level. Fluorescence units were significantly lower in F genes than in the other domains with a similar AT content. The transcriptional level of the domains categorized them was D>U>F. In addition, a comparison of 12 S. pneumoniae genome sequences showed a conservation of gene composition within U and D domains, and an extensive gene interchange in F domains. We tested the organization of chromosomal domains by measuring the relaxation-mediated transcription of eight insertions of a heterologous Ptccat cassette, two in each type of domain, showing that transcription depended on their chromosomal location. Moreover, transcription from the four promoters directing the five genes involved in supercoiling homeostasis, located either in U (gyrB), D (topA), or N (gyrA and parEC) domains was analyzed both in their chromosomal locations and in a replicating plasmid. Although expression from the chromosomal PgyrB and PtopA showed the expected domain regulation, their expression was down-regulated in the plasmid, which behaved as a D domain. However, both PparE and PgyrA carried their own regulatory signals, their topology-dependent expression being equivalent in the plasmid or in the chromosome. In PgyrA a DNA bend acted as a DNA supercoiling sensor. These results revealed that DNA topology functions as a general transcriptional regulator, superimposed upon other more specific regulatory mechanisms.

The pH-dependent expression of the urease operon in Streptococcus salivarius is mediated by CodY.

Urease gene expression in Streptococcus salivarius 57.I, a strain of one of the major alkali producers in the mouth, is induced by acidic pH and excess amounts of carbohydrate. Expression is controlled primarily at the transcriptional level from a promoter, pureI. Recent sequencing analysis revealed a CodY box located 2 bases 5' to the -35 element of pureI. Using continuous chemostat culture, transcription from pureI was shown to be repressed by CodY, and at pH 7 the repression was more pronounced than that in cells grown at pH 5.5 under both 20 and 100 mM glucose. The direct binding of CodY to pureI was demonstrated by electrophoretic mobility shift assay and chromatin immunoprecipitation (ChIP)-quantitative real-time PCR (qPCR). The result of ChIP-qPCR also confirmed that the regulation of CodY is indeed modulated by pH and the binding of CodY at neutral pH is further enhanced by a limited supply of glucose (20 mM). In the absence of CodY, the C-terminal domain of the RNA polymerase (RNAP) α subunit interacted with the AT tracks within the CodY box, indicating that CodY and RNAP compete for the same binding region. Such regulation could ensure optimal urease expression when the enzyme is most required, i.e., at an acidic growth pH with an excess amount of carbon nutrients.

A new tool for cloning and gene expression in Streptococcus pneumoniae

A new replicon suitable for cloning and gene expression was successfully introduced into Streptococcus pneumoniae. The non-integrative lactococcal vectors pIL253 (higher-copy) and pIL252 (lower-copy), which are based on the promiscuous theta-replicating plasmid pAMβ1, were established in pneumococcus. The stability and the small size of these plasmids, together with the presence of a helpful multi-cloning site make them a useful genetic tool for gene expression in this bacterium. The functionality of the system was tested by cloning and expressing the pneumococcal RNase R gene in pIL253. Full constitutive expression of the cloned gene was observed, clearly demonstrating that this plasmid can be used as an expression vector in S. pneumoniae. Moreover, gene expression can be regulated by the use of the lower- or higher-copy number vector versions. The existence of other replicative plasmids based on this family, which are also probably functional in pneumococcus, further broadens the cloning possibilities. We also show that S. pneumoniae cells can accommodate simultaneously pIL252 or pIL253 together with pLS1, a pMV158 derivative, which replicates via a rolling circle mechanism. This fact greatly increases the ability to manipulate this bacterium.The availability of a new family of replicative vectors for genetic manipulation in S. pneumoniae is an important contribution to the study of this pathogenic microorganism.

Cellobiose-Mediated Gene Expression in Streptococcus pneumoniae: A Repressor Function of the Novel GntR-Type Regulator BguR

The human pathogen Streptococcus pneumoniae has the ability to use the carbon- and energy source cellobiose due to the presence of a cellobiose-utilizing gene cluster (cel locus) in its genome. This system is regulated by the cellobiose-dependent transcriptional activator CelR, which has been previously shown to contribute to pneumococcal virulence. To get a broader understanding of the response of S. pneumoniae to cellobiose, we compared the pneumococcal transcriptome during growth on glucose as the main carbon source to that with cellobiose as the main carbon source. The expression of various carbon metabolic genes was altered, including a PTS operon (which we here denote as the bgu operon) that has high similarity with the cel locus. In contrast to the cel locus, the bgu operon is conserved in all sequenced strains of S. pneumoniae, indicating an important physiological function in the lifestyle of pneumococci. We next characterized the transcriptional regulation of the bgu operon in more detail. Its expression was increased in the presence of cellobiose, and decreased in the presence of glucose. A novel GntR-type transcriptional regulator (which we here denote as BguR) was shown to act as a transcriptional repressor of the bgu operon and its repressive effect was relieved in the presence of cellobiose. BguR-dependent repression was demonstrated to be mediated by a 20-bp DNA operator site (5′-AAAAATGTCTAGACAAATTT-3′) present in PbguA, as verified by promoter truncation experiments. In conclusion, we have identified a new cellobiose-responsive PTS operon, together with its transcriptional regulator in S. pneumoniae.

Construction of a plasmid vector based on the pMV158 replicon for cloning and inducible gene expression in Streptococcus pneumoniae

We report the construction of a plasmid vector designed for regulated gene expression in Streptococcus pneumoniae. The new vector, pLS1ROM, is based on the replicon of the streptococcal promiscuous rolling circle replication (RCR) plasmid pMV158. We inserted the controllable promoter P M of the S. pneumoniae malMP operon, followed by a multi-cloning site sequence aimed to facilitate the insertion of target genes. The expression from P M is negatively regulated by the transcriptional repressor MalR, which is released from the DNA operator sequence by growing the cells in maltose-containing media. To get a highly regulated expression of the target gene, MalR was provided in cis by inserting the malR gene under control of the constitutive P tet promoter, which in pMV158 directs expression of the tetL gene. To test the functionality of the system, we cloned the reporter gene gfp from Aequorea victoria, encoding the green fluorescent protein (GFP). Pneumococcal cells harboring the recombinant plasmid rendered GFP fluorescence in a maltose-dependent mode with undetectable background levels in the absence of the inducer. The new vector, pLS1ROM, exhibits full structural and segregational stability and constitutes a valuable tool for genetic manipulation and regulated gene expression in S. pneumoniae.

Dynamic changes of TrkB gene expression in Streptococcus pneumoniae meningitis after treatment with antibiotics and dexamethasone

Although more and more new potent antibiotics have been used, the incidence of neurological sequelae of Streptococcus pneumoniae meningitis has not improved in children over the last decade. The expression of TrkB mRNA, a receptor of brain-derived neurotrophic factor, is associated with the incidence of neurological sequelae of Streptococcus pneumoniae meningitis. Rats of 3 weeks old were used to construct a model of Streptococcus pneumoniae meningitis and served as normal controls. They were administered with antibiotics or antibiotics plus dexamethasone, respectively. The expression of the TrkB gene was detected in the brain by in situ hybridization. In the brains of Streptococcus pneumoniae inoculated rats, TrkB mRNA was significantly up-regulated after inoculation for 24 hours, and then down-regulated in a dose-dependent manner after treatment with antibiotics. This up-regulation was seen after treatment with antibiotics plus dexamethasone. TrkB mRNA expression was also observed in some infiltrating inflammatory cells. The results of the study support the hypothesis that TrkB signal transduction pathways might play an important role in Streptococcus pneumoniae meningitis, probably by protecting the brain from damage. The role of TrkB might be weakened after the treatment with antibiotics. Our findings suggest that targeting TrkB receptors might be a rational strategy for prevention of neurological sequelae caused by Streptococcus pneumoniae meningitis.

Distinct Time-Resolved Roles for Two Catabolite-Sensing Pathways during Streptococcus pyogenes Infection

Many Gram-positive pathogens link the expression of virulence genes to the presence of carbon source substrates using overlapping pathways for global control of carbon catabolite regulation. However, how these pathways are integrated to control the behavior of the transcriptome in time- and compartment-specific patterns is typically not well understood. In the present study, global transcriptome profiling was used to determine the extent to which glucose alters gene expression in Streptococcus pyogenes (group A streptococcus) and the contributions of the CcpA and LacD.1 catabolite control pathways to the regulation of this response in vitro. This analysis revealed that the expression of as many as 15% of the genes examined was regulated and that CcpA and LacD.1 together contribute to the regulation of 60% of this subset. However, numerous patterns were observed, including both CcpA- and LacD.1-specific and independent regulation, coregulation, and regulation of genes by these pathways independently of glucose. In addition, CcpA and LacD.1 had antagonistic effects on most coregulated genes. To resolve the roles of these regulators during infection, the expression of selected transcripts representative of different regulatory patterns was examined in a murine model of soft tissue infection. This revealed distinct patterns of misregulation with respect to time in CcpA(-) versus LacD.1(-) mutants. Taken together, these data support an important role for carbohydrate in the regulation of the transcriptome in tissue and suggest that the CcpA and LacD.1 pathways are organized to function at different times during the course of an infection.

Differential gene expression in Streptococcus pneumoniae in response to various iron sources

Iron is a critical co-factor for several enzymes and is known to regulate gene expression in many pathogens. Streptococcus pneumoniae (pneumococcus) normally colonizes the upper respiratory mucosa, which is an iron-restricted environment. In contrast, during bacteremia available iron from heme and non-heme proteins potentially increases. In iron-depleted medium pneumococcal strain TIGR4 showed reduced growth, however, addition of several physiological iron sources restored growth. Gene expression of selected known and putative pneumococcal virulence factors was analyzed by quantitative RT-PCR in response to iron sources in vitro and during colonization, pneumonia, and bacteremia in a mouse model. Change in mRNA levels relative to transcription in iron-depleted medium was reported. In presence of iron sources, transcription of cps4A, zmpA, pavA, hemolysin and a putative exfoliative toxin was significantly increased, but nanB was suppressed. Hemoglobin at physiological concentration repressed ply and pspA expression. Ferritin, an acute phase protein, increased expression of an iron ABC transporter and repressed expression of a bacterial non-heme iron-containing ferritin. Transcription of cps4A, nanB, hemolysin, and a putative exfoliative toxin were significantly up-regulated during pneumonia and bacteremia, while mRNA of pavA and non-heme ferritin were expressed at higher levels during pneumonia and carriage. An iron ABC transporter was most up-regulated during bacteremia, while pspA and ply were expressed only in pneumonia. Transcription of zmpA was elevated during both pneumonia and bacteremia. These findings suggest that a subset of virulence genes in pneumococci is differentially regulated in response to the quantity and form of iron sources available in a host.

Characterization of irvR , a Novel Regulator of the irvA -Dependent Pathway Required for Genetic Competence and Dextran-Dependent Aggregation in Streptococcus mutans

Previous studies identified irvA as a normally repressed but highly inducible transcription regulator capable of repressing mutacin I gene expression in Streptococcus mutans. In this study, we aimed to identify and characterize the regulator(s) responsible for repressing the expression of irvA. An uncharacterized open reading frame (SMU.1398) located immediately adjacent to irvA and annotated as a putative transcription repressor was identified as a likely candidate. The results of mutation studies confirmed that the expression of irvA was greatly increased in the SMU.1398 background. Mutation of SMU.1398 ("irvR") abolished genetic competence and reduced the expression of the late competence genes/operons comEA, comY, and dprA without affecting the expression of the known competence regulators comC, comED, or comX. In addition, irvR was found to be a potent negative regulator of dextran-dependent aggregation (DDAG) and gbpC expression. Each of these irvR mutant phenotypes could be rescued with a double mutation of irvA or complemented by introducing a wild-type copy of irvR on a shuttle vector. These data indicate that the repression of irvA is critically dependent upon irvR and that irvA repression is essential for the development of genetic competence and the proper control of DDAG in S. mutans.

Differential expression of key pneumococcal virulence genes in vivo

Few studies have examined in vivo virulence gene expression in Streptococcus pneumoniae. In this study, expression of key pneumococcal virulence genes cbpA, pspA, ply, psaA, cps2A, piaA, nanA and spxB in the nasopharynx, lungs and bloodstream of mice was investigated, following intranasal challenge with the serotype 2 strain D39. Bacterial RNA was extracted, linearly amplified and assayed by real-time RT-PCR. At 72 h, cbpA mRNA was present at higher levels in the nasopharynx and lungs than in the blood. At this time-point, the mRNAs for PspA and PiaA were most abundant in the nasopharynx, whereas no significant difference in gene expression between niches was observed for ply, psaA and cps2A. Both nanA and spxB mRNAs were present in higher amounts in the nasopharynx than in the lungs or blood. These findings illustrate the dynamic nature of pneumococcal virulence gene expression in vivo.

Cis-Acting elements that regulate the low-pH-inducible urease operon of Streptococcus salivarius.

Differential expression of the Streptococcus salivarius 57.I urease operon in response to pH is effected by repression of transcription from a proximal promoter, PUREI: To localize the cis-acting elements involved in the regulation of the urease operon, the intact promoter region and its derivatives were generated and fused to a promoterless chloramphenicol acetyltransferase (cat) gene. The promoter-cat fusions were established in the lacZ gene of S. salivarius by using a newly constructed integration vector. CAT-specific activities were examined in batch-grown cells at pH 7.5 and 5.5. The results indicated that a 21 bp region immediately 5' to the -35 element was required for efficient repression of PureI at neutral pH and that the 39 bp (-57 to -95) 5' to this region contained sequences required for optimal expression of PUREI: A potential secondary repressor-binding site was tentatively identified further upstream of the -35 element (-96 to -115). To further analyse the cis-acting elements, base changes were introduced into two AT-rich repeats within the primary repressor-binding site. One such derivative, S. salivarius M1, with five base substitutions immediately 5' to the -35 element, expressed 20-fold more CAT-specific activity at neutral pH than the strain carrying wild-type PureI-cat. Also, the pH sensitivity of strain M1 was greatly reduced, suggesting that this AT-rich region is crucial for repression of the urease operon. Deletion of three consecutive 15- or 16-base segments from -52 to -96 in the S. salivarius M1 background resulted in lower activities compared to strain M1, confirming the presence of sequences required for optimal expression of the operon. All of the PureI-cat fusions were also integrated into the gtfG gene of Streptococcus gordonii DL1, a non-ureolytic oral Streptococcus sp. Repression of PureI was observed at neutral pH in S. gordonii and the effects of the various mutations of the repressor-binding site largely paralleled those seen in S. salivarius, suggesting that the cis-elements may be a target for a global regulatory circuit that controls gene expression in streptococci in response to pH.

Integrational plasmids for the tetracycline-regulated expression of genes in Streptococcus pneumoniae.

Plasmids for the tetracycline regulated gene expression in Streptococcus pneumoniae have been developed. The plasmids were used for the tetracycline-dependent production of firefly luciferase in streptococci. The production of luciferase can be induced fivefold by the addition of tetracycline. By using two promoters of different strength and depending on the presence or absence of tetracycline, an 80-fold range of luciferase activities can be covered. The system was also used to construct strains that depend on the addition of tetracycline for the production of the A subunit of DNA gyrase, an essential streptococcal protein. The growth of such a strain depends on the addition of tetracycline to the medium. In the absence of tetracycline, the cells cease to grow and are not viable. The system presented in this report should be useful for the characterization of gene networks in S. pneumoniae. It especially allows one to study the function of essential genes that can not be investigated by standard knock-out techniques.

Comparative analysis of gene expression in Streptococcus pneumoniae and Lactococcus lactis

Comparative analysis of gene expression in Streptococcus pneumoniae and Lactococcus lactis

Tripartite streptokinase gene fusion vectors for gram-positive and gram-negative procaryotes

A specific 1,596 bp HincII fragment ('skc) from the chromosome of Streptococcus equisimilis contains an active streptokinase (SK) gene (skc) lacking, in addition to the expression signals, codons 1 through 39 of wild-type skc but retaining the remainder of the skc coding sequence together with the transcription terminator. Using this fragment as an indicator gene, we constructed two types of vectors which in appropriate hosts resulted in the synthesis of SK fusion proteins after insertional activation of 'skc. The first type are open reading frame (ORF) vectors in which 'skc was inserted into pUC18 out of frame with respect to lacZ', thus conferring an SK-negative phenotype. Any DNA fragments representing ORFs inserted between the lacZ' expression signals and 'skc such that the skc reading frame was restored resulted in the production of tripartite proteins which exhibited SK activity. The second type of vector, which functioned in both gram-positive and gram-negative bacteria, used the streptococcal speA expression and secretion signals in front of the ORF to activate 'skc insertionally. Using a large fragment from the chymosin gene as the target sequence, the usefulness of these vectors for studying foreign gene expression in streptococci as well as Escherichia coli was demonstrated.