Irfan Manzoor

The Regulation of the AdcR Regulon in Streptococcus pneumoniae Depends Both on Zn(2+)- and Ni(2+)-Availability.

By using a transcriptomic approach, we have elucidated the effect of Ni2+ on the global gene expression of S. pneumoniae D39 by identifying several differentially expressed genes/operons in the presence of a high extracellular concentration of Ni2+. The genes belonging to the AdcR regulon (adcRCBA, adcAII-phtD, phtA, phtB, and phtE) and the PsaR regulon (pcpA, prtA, and psaBCA) were highly upregulated in the presence of Ni2+. We have further studied the role of Ni2+ in the regulation of the AdcR regulon by using ICP-MS analysis, electrophoretic mobility shift assays and transcriptional lacZ-reporter studies, and demonstrate that Ni2+ is directly involved in the derepression of the AdcR regulon via the Zn2+-dependent repressor AdcR, and has an opposite effect on the expression of the AdcR regulon compared to Zn2+.

A Fast and Reliable Pipeline for Bacterial Transcriptome Analysis Case study: Serine-dependent Gene Regulation in Streptococcus pneumoniae

Gene expression and its regulation are very important to understand the behavior of cells under different conditions. Various techniques are used nowadays to study gene expression, but most are limited in terms of providing an overall picture of the expression of the whole transcriptome. DNA microarrays offer a fast and economic research technology, which gives a full overview of global gene expression and have a vast number of applications including identification of novel genes and transcription factor binding sites, characterization of transcriptional activity of the cells and also help in analyzing thousands of genes (in a single experiment). In the present study, the conditions for bacterial transcriptome analysis from cell harvest to DNA microarray analysis have been optimized. Taking into account the time, costs and accuracy of the experiments, this technology platform proves to be very useful and universally applicabale for studying bacterial transcriptomes. Here, we perform DNA microarray analysis with Streptococcus pneumoniae as a case-study by comparing the transcriptional responses of S. pneumoniae grown in the presence of varying L-serine concentrations in the medium. Total RNA was isolated by using a Macaloid method using an RNA isolation kit and the quality of RNA was checked by using an RNA quality check kit. cDNA was prepared using reverse transcriptase and the cDNA samples were labelled using one of two amine-reactive fluorescent dyes. Homemade DNA microarray slides were used for hybridization of the labelled cDNA samples and microarray data were analyzed by using a cDNA microarray data pre-processing framework (Microprep). Finally, Cyber-T was used to analyze the data generated using Microprep for the identification of statistically significant differentially expressed genes. Furthermore, in-house built software packages (PePPER, FIVA, DISCLOSE, PROSECUTOR, Genome2D) were used to analyze data.

GalR Acts as a Transcriptional Activator of galKT in the Presence of Galactose in Streptococcus pneumoniae

We explored the regulatory mechanism of Leloir pathway genes in Streptococcus pneumoniae D39. Here, we demonstrate that the expression of galKT is galactose dependent. By microarray analysis and quantitative RT-PCR, we further show the role of the transcriptional regulator GalR, present upstream of galKT, as a transcriptional activator of galKT in the presence of galactose. Moreover, we predict a 19-bp regulatory site (5′-GATAGTTTAGTAAAATTTT-3′) for the transcriptional regulator GalR in the promoter region of galK, which is also highly conserved in other streptococci. Growth comparison of D39 ΔgalK with the D39 wild type grown in the presence of galactose shows that galK is required for the proper growth of S. pneumoniae on galactose.

Co2+-dependent gene expression in Streptococcus pneumoniae: opposite effect of Mn2+ and Co2+ 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.

Maltose-Dependent Transcriptional Regulation of the mal Regulon by MalR in Streptococcus pneumoniae

The maltose regulon (mal regulon) has previously been shown to consist of the mal gene cluster (malMP, malXCD and malAR operons) in Streptococcus pneumoniae. In this study, we have further elucidated the complete mal regulon in S. pneumoniae D39 using microarray analyses and β-galactosidase assays. In addition to the mal gene cluster, the complete mal regulon of S. pneumoniae D39 consists of a pullulanase (PulA), a glucosidase (DexB), a glucokinase (RokB), a PTS component (PtsG) and an amylase (AmyA2). Our microarray studies and β-galactosidase assays further showed that the LacI-family transcriptional regulator MalR represses the expression of the mal regulon in the absence of maltose. Furthermore, the role of the pleiotropic transcriptional regulator CcpA in the regulation of the mal regulon in the presence of maltose was explored. Our microarray analysis with a ΔccpA strain showed that CcpA only represses the expression of the malXCD operon and the pulA gene in the presence of maltose. Hence, we extend the mal regulon now consisting of pulA, dexB, rokB, ptsG and amyA2 in addition to malMP, malXCD and malAR operons.

Ni2+-Dependent and PsaR-Mediated Regulation of the Virulence Genes pcpA, psaBCA, and prtA in Streptococcus pneumoniae

Previous studies have shown that the transcriptional regulator PsaR regulates the expression of the PsaR regulon consisting of genes encoding choline binding protein (PcpA), the extracellular serine protease (PrtA), and the Mn2+-uptake system (PsaBCA), in the presence of manganese (Mn2+), zinc (Zn2+), and cobalt (Co2+). In this study, we explore the Ni2+-dependent regulation of the PsaR regulon. We have demonstrated by qRT-PCR analysis, metal accumulation assays, β-galactosidase assays, and electrophoretic mobility shift assays that an elevated concentration of Ni2+ leads to strong induction of the PsaR regulon. Our ICP-MS data show that the Ni2+-dependent expression of the PsaR regulon is directly linked to high, cell-associated, concentration of Ni2+, which reduces the cell-associated concentration of Mn2+. In vitro studies with the purified PsaR protein showed that Ni2+ diminishes the Mn2+-dependent interaction of PsaR to the promoter regions of its target genes, confirming an opposite effect of Mn2+ and Ni2+ in the regulation of the PsaR regulon. Additionally, the Ni2+-dependent role of PsaR in the regulation of the PsaR regulon was studied by transcriptome analysis.

Fucose-Mediated Transcriptional Activation of the fcs Operon by FcsR in Streptococcus pneumoniae

In this study, we explore the impact of fucose on the transcriptome of S. pneumoniae D39. The expression of various genes and operons, including the fucose uptake PTS and utilization operon (fcs operon) was altered in the presence of fucose. By means of quantitative RT-PCR and β-galactosidase analysis, we demonstrate the role of the transcriptional regulator FcsR, present upstream of the fcs operon, as a transcriptional activator of the fcs operon. We also predict a 19-bp putative FcsR regulatory site (5′-ATTTGAACATTATTCAAGT-3′) in the promoter region of the fcs operon. The functionality of this predicted FcsR regulatory site was further confirmed by promoter-truncation experiments, where deletion of half of the FscR regulatory site or full deletion led to the abolition of expression of the fcs operon.

N-acetylglucosamine-Mediated Expression of nagA and nagB in Streptococcus pneumoniae

In this study, we have explored the transcriptomic response of Streptococcus pneumoniae D39 to N-acetylglucosamine (NAG). Transcriptome comparison of S. pneumoniae D39 wild-type grown in chemically defined medium (CDM) in the presence of 0.5% NAG to that grown in the presence of 0.5% glucose revealed elevated expression of many genes/operons, including nagA, nagB, manLMN, and nanP. We have further confirmed the NAG-dependent expression of nagA, nagB, manLMN, and nanP by β-galactosidase assays. nagA, nagB and glmS are putatively regulated by a transcriptional regulator NagR. We predicted the operator site of NagR (dre site) in PnagA, PnagB, and PglmS, which was further confirmed by mutating the predicted dre site in the respective promoters (nagA, nagB, and glmS). Growth comparison of ΔnagA, ΔnagB, and ΔglmS with the D39 wild-type demonstrates that nagA and nagB are essential for S. pneumoniae D39 to grow in the presence of NAG as a sole carbon source. Furthermore, deletion of ccpA shows that CcpA has no effect on the expression of nagA, nagB, and glmS in the presence of NAG in S. pneumoniae.

Rgg-Shp regulators are important for pneumococcal colonization and invasion through their effect on mannose utilization and capsule synthesis

Microbes communicate with each other by using quorum sensing (QS) systems and modulate their collective ‘behavior’ for in-host colonization and virulence, biofilm formation, and environmental adaptation. The recent increase in genome data availability reveals the presence of several putative QS sensing circuits in microbial pathogens, but many of these have not been functionally characterized yet, despite their possible utility as drug targets. To increase the repertoire of functionally characterized QS systems in bacteria, we studied Rgg144/Shp144 and Rgg939/Shp939, two putative QS systems in the important human pathogen Streptococcus pneumoniae. We find that both of these QS circuits are induced by short hydrophobic peptides (Shp) upon sensing sugars found in the respiratory tract, such as galactose and mannose. Microarray analyses using cultures grown on mannose and galactose revealed that the expression of a large number of genes is controlled by these QS systems, especially those encoding for essential physiological functions and virulence-related genes such as the capsular locus. Moreover, the array data revealed evidence for cross-talk between these systems. Finally, these Rgg systems play a key role in colonization and virulence, as deletion mutants of these QS systems are attenuated in the mouse models of colonization and pneumonia.

Interplay Between Capsule Expression and Uracil Metabolism in Streptococcus pneumoniae D39

Pyrimidine nucleotides play an important role in the biosynthesis of activated nucleotide sugars (NDP-sugars). NDP-sugars are the precursors of structural polysaccharides in bacteria, including capsule, which is a major virulence factor of the human pathogen S. pneumoniae. In this work, we identified a spontaneous non-reversible mutant of strain D39 that displayed a non-producing capsule phenotype. Whole-genome sequencing analysis of this mutant revealed several non-synonymous single base modifications, including in genes of the de novo synthesis of pyrimidines and in the −10 box of capsule operon promoter (Pcps). By directed mutagenesis we showed that the point mutation in Pcps was solely responsible for the drastic decrease in capsule expression. We also demonstrated that D39 subjected to uracil deprivation shows increased biomass and decreased Pcps activity and capsule amounts. Importantly, Pcps expression is further decreased by mutating the first gene of the de novo synthesis of pyrimidines, carA. In contrast, the absence of uracil from the culture medium showed no effect on the spontaneous mutant strain. Co-cultivation of the wild-type and the mutant strain indicated a competitive advantage of the spontaneous mutant (non-producing capsule) in medium devoid of uracil. We propose a model in that uracil may act as a signal for the production of different capsule amounts in S. pneumoniae.