Biology subjects

Lim, H. G.

Publications and source records attributed to Lim, H. G..

5 recordsLinked to original sources

bioRxiv2022

Revealing Pentose Catabolism in Pseudomonas putida

The Pseudomonas putida group in the Gammaproteobacteria has been intensively studied for bioremediation and plant growth promotion. Members of this group have recently emerged as promising hosts to convert intermediates derived from plant biomass to biofuels and biochemicals. However, most strains of P. putida cannot metabolize pentose sugars derived from hemicellulose. Here we describe three isolates that provide a broader view of the pentose sugar catabolism in the P. putida group. One of these isolates clusters with the well-characterized P. alloputida KT2440 (strain BP6); the second isolate clustered with plant growth-promoting strain P. putida W619 (strain M2), while the third isolate represents a new species in the group (strain BP8). Each of these isolates possessed homologous genes for oxidative xylose catabolism (xylDXA) and a potential xylonate transporter. Strain M2 grew on arabinose and had genes for oxidative arabinose catabolism (araDXA). A CRISPRi system was developed for strain M2 and identified conditionally essential genes for xylose growth. A glucose dehydrogenase was found to be responsible for initial oxidation of xylose and arabinose in strain M2. These isolates have illuminated inherent diversity in pentose catabolism in the P. putida group and may provide alternative hosts for biomass conversion. Originality-Significance StatementMembers of the Pseudomonas putida group are intensively studied for their role in plant growth promotion and biomass conversion. Despite this interest, the scope of pentose oxidation, key sugars in plant biomass, in this group is not known. Here, we report targeted isolation of members of the P. putida group that grow by xylose and arabinose oxidation. Using a combined genomic and proteomic approach, we identify gene products involved in pentose oxidation and identify conditionally essential genes for xylose oxidation using a CRISPRi gene repression approach. This work describes a targeted isolation and analysis strategy that may applied for many microbial groups of industrial and agricultural interest.

microbiology↗

bioRxiv2022

Elucidation of independently modulated genes in Streptococcus pyogenes reveals carbon sources that control its expression of hemolytic toxins

Streptococcus pyogenes can cause a wide variety of acute infections throughout the body of its human host. The underlying transcriptional regulatory network (TRN) is responsible for altering the physiological state of the bacterium to adapt to each host environment. Consequently, an in-depth understanding the comprehensive dynamics of its TRN could inform new therapeutic strategies. Here, we compiled 116 existing high-quality RNA-seq data sets of S. pyogenes serotype M1, and estimated the TRN structure in a top-down fashion by performing independent component analysis (ICA). The algorithm computed 42 independently modulated sets of genes (iModulons). Four iModulons contained nga-ifs-slo virulence-related operon, which allowed us to identify carbon sources that control its expression. In particular, dextrin utilization upregulated nga-ifs-slo operon by activation of two-component regulatory system CovRS-related iModulons, and changed bacterial hemolytic activity compared to glucose or maltose utilization. Finally, we show that the iModulon-based TRN structure can be used to simplify interpretation of noisy bacterial transcriptome at the infection site.

microbiology↗

bioRxiv2022

Machine-learning from Pseudomonas putida transcriptomes reveals its transcriptional regulatory network

Bacterial gene expression is orchestrated by numerous transcription factors (TFs). Elucidating how gene expression is regulated is fundamental to understanding bacterial physiology and engineering it for practical use. In this study, a machine-learning approach was applied to uncover the genome-scale transcriptional regulatory network (TRN) in Pseudomonas putida, an important organism for bioproduction. We performed independent component analysis of a compendium of 321 high-quality gene expression profiles, which were previously published or newly generated in this study. We identified 84 groups of independently modulated genes (iModulons) that explain 75.7% of the total variance in the compendium. With these iModulons, we (i) expand our understanding of the regulatory functions of 39 iModulon associated TFs (e.g., HexR, Zur) by systematic comparison with 1,993 previously reported TF-gene interactions; (ii) outline transcriptional changes after the transition from the exponential growth to stationary phases; (iii) capture group of genes required for utilizing diverse carbon sources and increased stationary response with slower growth rates; (iv) unveil multiple evolutionary strategies of transcriptome reallocation to achieve fast growth rates; and (v) define an osmotic stimulon, which includes the Type VI secretion system, as coordination of multiple iModulon activity changes. Taken together, this study provides the first quantitative genome-scale TRN for P. putida and a basis for a comprehensive understanding of its complex transcriptome changes in a variety of physiological states.

systems biology↗

bioRxiv2021

A systems approach discovers the role and characteristics of seven LysR type transcription factors in Escherichia coli

Although Escherichia coli K-12 strains represent perhaps the best known model bacteria, we do not know the identity or functions of all of their transcription factors (TFs). It is now possible to systematically discover the physiological function of TFs in E. coli BW25113 using a set of synergistic methods; including ChIP-exo, growth phenotyping, conserved gene clustering, and transcriptome analysis. Among 47 LysR-type TFs (LTFs) found on the E. coli K-12 genome, many regulate nitrogen source utilization or amino acid metabolism. However, 19 LTFs remain unknown. In this study, we elucidated the regulation of seven of these 19 LTFs: YbdO, YbeF, YgfI, YiaU, YneJ, YcaN, YbhD. We show that: 1) YbdO regulation has an effect on bacterial growth at low pH with citrate supplementation. YbdO is a repressor of the ybdNM operon and is implicated in the regulation of citrate lyase genes (citCDEFG); 2) YgfI activates the dhaKLM operon that encodes the phosphotransferase system involved in glycerol and dihydroxyacetone utilization; 3) YiaU regulates the yiaT gene encoding an outer membrane protein, and waaPSBOJYZU operon is also important in determining cell density at the stationary phase; 4) YneJ, re-named here as PtrR, directly regulates the expression of the succinate-semialdehyde dehydrogenase, Sad (also known as YneI), and is a predicted regulator of fnrS (a small RNA molecule). PtrR is important for bacterial growth in the presence of L-glutamate and putrescine as nitrogen sources; and 5) YbhD and YcaN regulate adjacent y-genes on the genome and YbeF is involved in flagella gene regulation. We have thus established the functions for four LTFs and identified the target genes for three LTFs. IMPORTANCEThe reconstruction of the transcriptional regulatory network (TRN) is important for gram-negative bacteria such as E. coli. LysR-type TFs are abundant in Enterobacteria, but many LTF functions still remain unknown. Here we report putative functions of uncharacterized TFs based on multi-omics data related to L-threonine, L-glutamate, and putrescine utilization. Amino acids (AAs) and polyamines are important sources of nitrogen for many microorganisms, but the increase in one amino acid or putrescine concentration in a minimal medium also induces stress. Although polyamine metabolism has been studied, the TRN that controls the putrescine (Ptr) and AA utilization at minimal medium conditions are still poorly understood. The function of previously uncharacterized transcriptional regulators YbdO, YgfI, and YneJ (PtrR) were identified in Escherichia coli. PtrR is important for Ptr and L-glutamate utilization, while YgfI transcriptional regulation was found to be important for growth on L-threonine and glycerol as a carbon source.

systems biology↗

bioRxiv2021

Unraveling the functions of uncharacterized transcription factors in Escherichia coli using ChIP-exo

Bacteria regulate gene expression to adapt to changing environments through transcriptional regulatory networks (TRNs). Although extensively studied, no TRN is fully characterized since the identity and activity of all the transcriptional regulators that comprise a TRN are not known. Here, we experimentally evaluate 40 uncharacterized proteins in Escherichia coli K-12 MG1655, which were computationally predicted to be transcription factors (TFs). First, we used a multiplexed ChIP-exo assay to characterize genome-wide binding sites for these candidate TFs; 34 of them were found to be DNA-binding protein. We then compared the relative location between binding sites and RNA polymerase (RNAP). We found 48% (283/588) overlap between the TFs and RNAP. Finally, we used these data to infer potential functions for 10 of the 34 TFs with validated DNA binding sites and consensus binding motifs. These TFs were found to have various roles in regulating primary cellular processes in E. coli. Taken together, this study: (1) significantly expands the number of confirmed TFs, close to the estimated total of about 280 TFs; (2) predicts the putative functions of the newly discovered TFs, and (3) confirms the functions of representative TFs through mutant phenotypes.

systems biology↗