Co-component signal transduction systems: Fast-evolving virulence regulation cassettes discovered in enteric bacteria.

Bacterial signal transduction systems sense changes in the environment and transmit these signals to control cellular responses. The simplest one-component signal transduction systems include an input sensor domain and an output response domain encoded in a single protein chain. Alternatively, two-component signal transduction systems transmit signals by phosphorelay between input and output domains from separate proteins. The membrane-tethered periplasmic bile acid sensor that activates the Vibrio parahaemolyticus type III secretion system adopts an obligate heterodimer of two proteins encoded by partially overlapping VtrA and VtrC genes. This co-component signal transduction system binds bile acid using a lipocalin-like domain in VtrC and transmits the signal through the membrane to a cytoplasmic DNA-binding transcription factor in VtrA. Using the domain and operon organization of VtrA/VtrC, we identify a fast-evolving superfamily of co-component systems in enteric bacteria. Accurate machine learning­based fold predictions for the candidate co-components support their homology in the twilight zone of rapidly evolving sequences and provide mechanistic hypotheses about previously unrecognized lipid-sensing functions.

Microarray profiling and identification of core promoter sequence in Gordonia.

Gordonia are Gram-positive bacteria which have immense biotechnological potential. Genomes of several Gordonia spp. have been sequenced but a detailed analysis of the differentially expressed genes during growth, the promoters which drive their expression and the information on the core promoter sequence is lacking. Here, we report the identification of core promoter sequence in Gordonia sp. IITR100. The GC content of the promoters was found to be within a range of 62-65%. The 5'-UTR length in the genes was also analysed and about 56% promoters were found to have long 5'-UTR. The functionality of the promoters was validated by microarray profiling. Based on the differential expression of genes, two growth phase dependent promoters PdsbA and Pglx were isolated and analysed. They add to the existing repertoire of the promoters functional in both Gram-negative and Gram-positive bacteria. Our results suggest that the core promoter sequence identified is conserved in members of Gordonia spp. and is similar to that of other members of Actinobacteria.

Strong synthetic stationary phase promoter-based gene expression system for Escherichia coli.

The Gram-negative bacterium Escherichia coli has been the work horse for recombinant protein production since the past several years. However, most of the gene expression systems used either require expensive inducers or exhibit low strength. In the present study, we have generated a strong promoter by repeated rounds of random mutagenesis in a stationary phase promoter isolated from Gordonia sp. IITR100. The promoter activity increased 16-fold as compared to the wild-type promoter. The resultant synthetic promoter showed ß-galactosidase activities of ~16,000 Miller units which is comparable to the strong T7 promoter ~13,000 Miller units. The amount of LacZ produced by the synthetic promoter was found to be active for several days in stationary phase. The advantage of this synthetic promoter over T7 promoter includes its stationary phase auto-inducibility thereby saving the cost of addition of inducers. Expression of GFPuv was observed in all the cells of E. coli due to the absence of requirement of inducer. A general-purpose vector containing the synthetic promoter with an MCS ready for use has been developed in the study. It has also been used to demonstrate the production of two heterologous proteins.

Membrane-localized expression, production and assembly of Vibrio parahaemolyticus T3SS2 provides evidence for transertion.

It has been proposed that bacterial membrane proteins may be synthesized and inserted into the membrane by a process known as transertion, which involves membrane association of their encoding genes, followed by coupled transcription, translation and membrane insertion. Here, we provide evidence supporting that the pathogen Vibrio parahaemolyticus uses transertion to assemble its type III secretion system (T3SS2), to inject virulence factors into host cells. We propose a two-step transertion process where the membrane-bound co-component receptor (VtrA/VtrC) is first activated by bile acids, leading to membrane association and expression of its target gene, vtrB, located in the T3SS2 pathogenicity island. VtrB, the transmembrane transcriptional activator of T3SS2, then induces the localized expression and membrane assembly of the T3SS2 structural components and its effectors. We hypothesize that the proposed transertion process may be used by other enteric bacteria for efficient assembly of membrane-bound molecular complexes in response to extracellular signals.

Characterization of divergent promoters PmaiA and Phyd from Gordonia: Co-expression and regulation by CRP.

Divergent promoters are often responsible for controlling gene expression of related genes of the same pathway or for coordinating regulation at different time points. There are relatively few reports on characterization of divergent promoters in bacteria. In the present study, microarray profiling was carried out to analyze gene expression during growth of Gordonia sp. IITR100, which led to the identification of 35 % of adjacent gene candidates that are divergently transcribed. We focus here on the in-depth characterization of one such pair of genes. Two divergent promoters, PmaiA and Phyd, drive the expression of genes encoding maleate cis-trans isomerase (maiA) and hydantoinase (hyd), respectively. Our findings reveal asymmetric promoter activity with higher activity in the reverse orientation (Phyd) as compared to the forward orientation (PmaiA). Minimal promoter region for each orientation was identified by deletion mapping. Deletion of a 5'-untranslated region of each gene resulted in an increase in promoter activity. A putative binding site for CRP (Catabolite Repressor Protein) transcription regulator was also identified in the 80 bp common regulatory region between the -35 hexamers of the two promoters. The results of this study suggest that CRP-mediated repression of PmaiA occurs only in the cells grown in glucose. Phyd, on the other hand, is not repressed by CRP. However, deletion of the CRP binding site located between -95 to -110 upstream to the transcription start site of the maiA gene resulted in increased activity of PmaiA and decreased activity of Phyd. A single CRP binding site, therefore, affects the two promoters differently.

Characterization of Zymomonas mobilis promoters that are functional in Escherichia coli.

Zymomonas mobilis ZM4 is a gram-negative, facultative anaerobic, natural ethanologenic bacterium used in industrial production of bio-products. For expression of genes, promoters are required. However, most of the promoters reported from Z. mobilis poorly function in Escherichia coli. This makes the process of expression and screening labor-intensive. In the present study, we compared the strengths of two Z. mobilis promoters, Pchap and Ppap, which drive the expression of chaperonin and phosphatase PAP2 family protein, respectively, with Ptac promoter. In E. coli, the Ptac promoter was found to be the strongest followed by Ppap and Ppdc, while in Z. mobilis, Ppdc was found to be the strongest and Pchap the weakest promoter. Further characterization of the promoters was done by cloning the gfpuv gene which expresses the green fluorescent protein, under their control and measuring the fluorescence of the E. coli transformants. The activity of these promoters was also studied at different pH (pH 5, 7 and 9) and different temperatures (30°C, 37°C and 42°C) in exponential and stationary phases. Both Pchap and Ppap promoters showed higher activity in stationary phase than in exponential phase. Since the promoters were active at all temperatures and pH studied, they can be used for gene expression in E. coli under desired environmental conditions.

Molecular Basis of Stationary Phase Survival and Applications.

Stationary phase is the stage when growth ceases but cells remain metabolically active. Several physical and molecular changes take place during this stage that makes them interesting to explore. The characteristic proteins synthesized in the stationary phase are indispensable as they confer viability to the bacteria. Detailed knowledge of these proteins and the genes synthesizing them is required to understand the survival in such nutrient deprived conditions. The promoters, which drive the expression of these genes, are called stationary phase promoters. These promoters exhibit increased activity in the stationary phase and less or no activity in the exponential phase. The vectors constructed based on these promoters are ideal for large-scale protein production due to the absence of any external inducers. A number of recombinant protein production systems have been developed using these promoters. This review describes the stationary phase survival of bacteria, the promoters involved, their importance, regulation, and applications.

Draft Genome Sequence of a Biodesulfurizing Bacterium, Gordonia sp. Strain IITR100.

We report here the whole-genome sequence of a biodesulfurizing bacterium, Gordonia sp. strain IITR100. The bacterium has the unique ability to desulfurize both aliphatic and aromatic organosulfurs. The draft genome sequence will provide insights into the various genes and regulators involved in biodesulfurization and other catabolic pathways.

Genome-wide analysis of heat shock proteins in C4 model, foxtail millet identifies potential candidates for crop improvement under abiotic stress.

Heat shock proteins (HSPs) perform significant roles in conferring abiotic stress tolerance to crop plants. In view of this, HSPs and their encoding genes were extensively characterized in several plant species; however, understanding their structure, organization, evolution and expression profiling in a naturally stress tolerant crop is necessary to delineate their precise roles in stress-responsive molecular machinery. In this context, the present study has been performed in C4 panicoid model, foxtail millet, which resulted in identification of 20, 9, 27, 20 and 37 genes belonging to SiHSP100, SiHSP90, SiHSP70, SiHSP60 and SisHSP families, respectively. Comprehensive in silico characterization of these genes followed by their expression profiling in response to dehydration, heat, salinity and cold stresses in foxtail millet cultivars contrastingly differing in stress tolerance revealed significant upregulation of several genes in tolerant cultivar. SisHSP-27 showed substantial higher expression in response to heat stress in tolerant cultivar, and its over-expression in yeast system conferred tolerance to several abiotic stresses. Methylation analysis of SiHSP genes suggested that, in susceptible cultivar, higher levels of methylation might be the reason for reduced expression of these genes during stress. Altogether, the study provides novel clues on the role of HSPs in conferring stress tolerance.

Integrative analysis and expression profiling of secondary cell wall genes in C4 biofuel model Setaria italica reveals targets for lignocellulose bioengineering.

Several underutilized grasses have excellent potential for use as bioenergy feedstock due to their lignocellulosic biomass. Genomic tools have enabled identification of lignocellulose biosynthesis genes in several sequenced plants. However, the non-availability of whole genome sequence of bioenergy grasses hinders the study on bioenergy genomics and their genomics-assisted crop improvement. Foxtail millet (Setaria italica L.; Si) is a model crop for studying systems biology of bioenergy grasses. In the present study, a systematic approach has been used for identification of gene families involved in cellulose (CesA/Csl), callose (Gsl) and monolignol biosynthesis (PAL, C4H, 4CL, HCT, C3H, CCoAOMT, F5H, COMT, CCR, CAD) and construction of physical map of foxtail millet. Sequence alignment and phylogenetic analysis of identified proteins showed that monolignol biosynthesis proteins were highly diverse, whereas CesA/Csl and Gsl proteins were homologous to rice and Arabidopsis. Comparative mapping of foxtail millet lignocellulose biosynthesis genes with other C4 panicoid genomes revealed maximum homology with switchgrass, followed by sorghum and maize. Expression profiling of candidate lignocellulose genes in response to different abiotic stresses and hormone treatments showed their differential expression pattern, with significant higher expression of SiGsl12, SiPAL2, SiHCT1, SiF5H2, and SiCAD6 genes. Further, due to the evolutionary conservation of grass genomes, the insights gained from the present study could be extrapolated for identifying genes involved in lignocellulose biosynthesis in other biofuel species for further characterization.

Global analysis of WRKY transcription factor superfamily in Setaria identifies potential candidates involved in abiotic stress signaling.

Transcription factors (TFs) are major players in stress signaling and constitute an integral part of signaling networks. Among the major TFs, WRKY proteins play pivotal roles in regulation of transcriptional reprogramming associated with stress responses. In view of this, genome- and transcriptome-wide identification of WRKY TF family was performed in the C4model plants, Setaria italica (SiWRKY) and S. viridis (SvWRKY), respectively. The study identified 105 SiWRKY and 44 SvWRKY proteins that were computationally analyzed for their physicochemical properties. Sequence alignment and phylogenetic analysis classified these proteins into three major groups, namely I, II, and III with majority of WRKY proteins belonging to group II (53 SiWRKY and 23 SvWRKY), followed by group III (39 SiWRKY and 11 SvWRKY) and group I (10 SiWRKY and 6 SvWRKY). Group II proteins were further classified into 5 subgroups (IIa to IIe) based on their phylogeny. Domain analysis showed the presence of WRKY motif and zinc finger-like structures in these proteins along with additional domains in a few proteins. All SiWRKY genes were physically mapped on the S. italica genome and their duplication analysis revealed that 10 and 8 gene pairs underwent tandem and segmental duplications, respectively. Comparative mapping of SiWRKY and SvWRKY genes in related C4 panicoid genomes demonstrated the orthologous relationships between these genomes. In silico expression analysis of SiWRKY and SvWRKY genes showed their differential expression patterns in different tissues and stress conditions. Expression profiling of candidate SiWRKY genes in response to stress (dehydration and salinity) and hormone treatments (abscisic acid, salicylic acid, and methyl jasmonate) suggested the putative involvement of SiWRKY066 and SiWRKY082 in stress and hormone signaling. These genes could be potential candidates for further characterization to delineate their functional roles in abiotic stress signaling.