Eugene P. Kennedy’s Legacy: Defining Bacterial Phospholipid Pathways and Function

In the 1950’s and 1960’s Eugene P. Kennedy laid out the blueprint for phospholipid biosynthesis in somatic cells and Escherichia coli, which have been coined the Kennedy Pathways for phospholipid biosynthesis. His research group continued to make seminal contributions in the area of phospholipids until his retirement in the early 1990’s. During these years he mentored many young scientists that continued to build on his early discoveries and who also mentored additional scientists that continue to make important contributions in areas related to phospholipids and membrane biogenesis. This review will focus on the initial E. coli Kennedy Pathways and how his early contributions have laid the foundation for our current understanding of bacterial phospholipid genetics, biochemistry and function as carried on by his scientific progeny and others who have been inspired to study microbial phospholipids.

Functional Studies of [FeFe] Hydrogenase Maturation in an Escherichia coli Biosynthetic System

10.1128/jb.188.6.2163-2172.2006 ◽

2006 ◽

Vol 188 (6) ◽

pp. 2163-2172 ◽

Cited By ~ 212

Author(s):

Paul W. King ◽

Matthew C. Posewitz ◽

Maria L. Ghirardi ◽

Michael Seibert

Keyword(s):

Escherichia Coli ◽

Catalytic Domain ◽

Expression System ◽

Iron Sulfur Cluster ◽

Sulfur Cluster ◽

E Coli ◽

Functional Studies ◽

Hydrogenase Maturation ◽

Iron Sulfur ◽

ABSTRACT Maturation of [FeFe] hydrogenases requires the biosynthesis and insertion of the catalytic iron-sulfur cluster, the H cluster. Two radical S-adenosylmethionine (SAM) proteins proposed to function in H cluster biosynthesis, HydEF and HydG, were recently identified in the hydEF-1 mutant of the green alga Chlamydomonas reinhardtii (M. C. Posewitz, P. W. King, S. L. Smolinski, L. Zhang, M. Seibert, and M. L. Ghirardi, J. Biol. Chem. 279:25711-25720, 2004). Previous efforts to study [FeFe] hydrogenase maturation in Escherichia coli by coexpression of C. reinhardtii HydEF and HydG and the HydA1 [FeFe] hydrogenase were hindered by instability of the hydEF and hydG expression clones. A more stable [FeFe] hydrogenase expression system has been achieved in E. coli by cloning and coexpression of hydE, hydF, and hydG from the bacterium Clostridium acetobutylicum. Coexpression of the C. acetobutylicum maturation proteins with various algal and bacterial [FeFe] hydrogenases in E. coli resulted in purified enzymes with specific activities that were similar to those of the enzymes purified from native sources. In the case of structurally complex [FeFe] hydrogenases, maturation of the catalytic sites could occur in the absence of an accessory iron-sulfur cluster domain. Initial investigations of the structure and function of the maturation proteins HydE, HydF, and HydG showed that the highly conserved radical-SAM domains of both HydE and HydG and the GTPase domain of HydF were essential for achieving biosynthesis of active [FeFe] hydrogenases. Together, these results demonstrate that the catalytic domain and a functionally complete set of Hyd maturation proteins are fundamental to achieving biosynthesis of catalytic [FeFe] hydrogenases.

Genetic Organization of the Vibrio harveyi dnaA Gene Region and Analysis of the Function of the V. harveyi DnaA Protein in Escherichia coli

10.1128/jb.184.9.2533-2538.2002 ◽

2002 ◽

Vol 184 (9) ◽

pp. 2533-2538 ◽

Cited By ~ 8

Author(s):

Dvora Berenstein ◽

Kirsten Olesen ◽

Christian Speck ◽

Ole Skovgaard

Keyword(s):

Escherichia Coli ◽

Promoter Region ◽

Vibrio Harveyi ◽

Gene Region ◽

Chromosomal Dna ◽

Dnaa Gene ◽

E Coli ◽

Dnaa Protein ◽

Dam Methylase ◽

ABSTRACT The Vibrionaceae family is distantly related to Enterobacteriaceae within the group of bacteria possessing the Dam methylase system. We have cloned, sequenced, and analyzed the dnaA gene region of Vibrio harveyi and found that although the organization of the V. harveyi dnaA region differs from that of Escherichia coli, the expression of both genes is autoregulated and ATP-DnaA binds cooperatively to ATP-DnaA boxes in the dnaA promoter region. The DnaA proteins of V. harveyi and E. coli are interchangeable and function nearly identically in controlling dnaA transcription and the initiation of chromosomal DNA replication despite the evolutionary distance between these bacteria.

Identification and Characterization of Genes Required for 5-Hydroxyuridine Synthesis in Bacillus subtilis and Escherichia coli tRNA

10.1128/jb.00433-19 ◽

2019 ◽

Vol 201 (20) ◽

Cited By ~ 7

Author(s):

Charles T. Lauhon

Keyword(s):

Escherichia Coli ◽

Bacillus Subtilis ◽

Genomic Context ◽

Similar Reduction ◽

Content Type ◽

E Coli ◽

Wobble Position ◽

Fertile Ground ◽

ABSTRACT In bacteria, tRNAs that decode 4-fold degenerate family codons and have uridine at position 34 of the anticodon are typically modified with either 5-methoxyuridine (mo5U) or 5-methoxycarbonylmethoxyuridine (mcmo5U). These modifications are critical for extended recognition of some codons at the wobble position. Whereas the alkylation steps of these modifications have been described, genes required for the hydroxylation of U34 to give 5-hydroxyuridine (ho5U) remain unknown. Here, a number of genes in Escherichia coli and Bacillus subtilis are identified that are required for wild-type (wt) levels of ho5U. The yrrMNO operon is identified in B. subtilis as important for the biosynthesis of ho5U. Both yrrN and yrrO are homologs to peptidase U32 family genes, which includes the rlhA gene required for ho5C synthesis in E. coli. Deletion of either yrrN or yrrO, or both, gives a 50% reduction in mo5U tRNA levels. In E. coli, yegQ was found to be the only one of four peptidase U32 genes involved in ho5U synthesis. Interestingly, this mutant shows the same 50% reduction in (m)cmo5U as that observed for mo5U in the B. subtilis mutants. By analyzing the genomic context of yegQ homologs, the ferredoxin YfhL is shown to be required for ho5U synthesis in E. coli to the same extent as yegQ. Additional genes required for Fe-S biosynthesis and biosynthesis of prephenate give the same 50% reduction in modification. Together, these data suggest that ho5U biosynthesis in bacteria is similar to that of ho5C, but additional genes and substrates are required for complete modification. IMPORTANCE Modified nucleotides in tRNA serve to optimize both its structure and function for accurate translation of the genetic code. The biosynthesis of these modifications has been fertile ground for uncovering unique biochemistry and metabolism in cells. In this work, genes that are required for a novel anaerobic hydroxylation of uridine at the wobble position of some tRNAs are identified in both Bacillus subtilis and Escherichia coli. These genes code for Fe-S cluster proteins, and their deletion reduces the levels of the hydroxyuridine by 50% in both organisms. Additional genes required for Fe-S cluster and prephenate biosynthesis and a previously described ferredoxin gene all display a similar reduction in hydroxyuridine levels, suggesting that still other genes are required for the modification.

Contribution of SOS Genes to H2O2-induced Apoptosis-like Death in Escherichia Coli

10.21203/rs.3.rs-520509/v1 ◽

2021 ◽

Author(s):

Heesu Kim ◽

Dong Gun Lee

Keyword(s):

Oxidative Stress ◽

Escherichia Coli ◽

Treatment Strategies ◽

Sos Response ◽

Bacterial Dna ◽

E Coli ◽

New Treatment ◽

Induced Apoptosis ◽

And Function ◽

The Relationship

Abstract Hydrogen peroxide (H2O2) is a debriding agent that damages the microbial structure and function by generating various reactive oxygen species (ROS). H2O2-produced hydroxyl radical (OH∙) also exert oxidative stress on microorganisms. The spread of antibiotic resistance in bacteria is a serious issue worldwide, and greater efforts are needed to identify and characterize novel antibacterial mechanisms to develop new treatment strategies. Therefore, this study aimed to clarify the relationship between H2O2 and Escherichia coli and to elucidate a novel antibacterial mechanism(s) of H2O2. Following H2O2 exposure, increased levels of 8-hydroxyldeoxyguanosine and malondialdehyde indicated that H2O2 accelerates oxidation of bacterial DNA and lipids in E. coli. As oxidative damage worsened, the SOS response was triggered. Cell division arrest and resulting filamentation were identified in cells, indicating that LexA was involved in DNA replication. It was also verified that RecA, a representative SOS gene, helps self-cleavage of LexA and acts as a bacterial caspase-like protein. Our findings also showed that dinF is essential to preserve E. coli from H2O2-induced ROS, and furthermore, demonstrated that H2O2-induced SOS response and SOS genes participate differently in guarding E. coli from oxidative stress. As an extreme SOS response is considered apoptosis-like death (ALD) in bacteria, additional experiments were performed to examine the characteristics of ALD. DNA fragmentation and membrane depolarization appeared in H2O2-treated cells, suggesting that H2O2 causes ALD in E. coli. In conclusion, our investigations revealed that ALD is a novel antibacterial mode of action(s) of H2O2 with important contributions from SOS genes.

CS5 Pilus Biosynthesis Genes from Enterotoxigenic Escherichia coli O115:H40

10.1128/jb.181.18.5847-5851.1999 ◽

1999 ◽

Vol 181 (18) ◽

pp. 5847-5851 ◽

Cited By ~ 3

Author(s):

Thomas G. Duthy ◽

Lothar H. Staendner ◽

Paul A. Manning ◽

Michael W. Heuzenroeder

Keyword(s):

Escherichia Coli ◽

Dna Sequences ◽

Surface Expression ◽

Open Reading Frames ◽

Enterotoxigenic Escherichia Coli ◽

Cell Surface Expression ◽

E Coli ◽

Pilus Biogenesis ◽

K 12 ◽

ABSTRACT We have sequenced the entire region of DNA required for the biosynthesis of CS5 pili from enterotoxigenic Escherichia coli O115:H40 downstream of the major subunit gene, designatedcsfA (for coli surface factor five A). Five more open reading frames (ORFs) (csfB, csfC,csfE, csfF, and csfD) which are transcribed in the same direction as the major subunit and are flanked by a number of insertion sequence regions have been identified. T7 polymerase-mediated overexpression of the cloned csf ORFs confirmed protein sizes based on the DNA sequences that encode them. The expression of only the csf region in E. coli K-12 resulted in the hemagglutination of human erythrocytes and the cell surface expression of CS5 pili, suggesting that the cluster contains all necessary information for CS5 pilus biogenesis and function.

Requirements for Conversion of the Na+-Driven Flagellar Motor of Vibrio cholerae to the H+-Driven Motor of Escherichia coli

10.1128/jb.182.15.4234-4240.2000 ◽

2000 ◽

Vol 182 (15) ◽

pp. 4234-4240 ◽

Cited By ~ 64

Author(s):

Khoosheh K. Gosink ◽

Claudia C. Häse

Keyword(s):

Escherichia Coli ◽

Vibrio Cholerae ◽

Proton Translocation ◽

Specific Inhibitor ◽

The Other ◽

E Coli ◽

Alkaline Conditions ◽

Flagellar Assembly ◽

And Function ◽

Hybrid Motor

ABSTRACT Bacterial flagella are powered by a motor that converts a transmembrane electrochemical potential of either H+ or Na+ into mechanical work. In Escherichia coli, the MotA and MotB proteins form the stator and function in proton translocation, whereas the FliG protein is located on the rotor and is involved in flagellar assembly and torque generation. The sodium-driven polar flagella of Vibrio species contain homologs of MotA and MotB, called PomA and PomB, and also contain two other membrane proteins called MotX and MotY, which are essential for motor rotation and that might also function in ion conduction. Deletions inpomA, pomB, motX, ormotY in Vibrio cholerae resulted in a nonmotile phenotype, whereas deletion of fliG gave a nonflagellate phenotype. fliG genes on plasmids complementedfliG-null strains of the parent species but notfliG-null strains of the other species. FliG-null strains were complemented by chimeric FliG proteins in which the C-terminal domain came from the other species, however, implying that the C-terminal part of FliG can function in conjunction with the ion-translocating components of either species. A V. cholerae strain deleted of pomA, pomB,motX, and motY became weakly motile when theE. coli motA and motB genes were introduced on a plasmid. Like E. coli, but unlike wild-type V. cholerae, motility of some V. cholerae strains containing the hybrid motor was inhibited by the protonophore carbonyl cyanide m-chlorophenylhydrazone under neutral as well as alkaline conditions but not by the sodium motor-specific inhibitor phenamil. We conclude that the E. coli proton motor components MotA and MotB can function in place of the motor proteins ofV. cholerae and that the hybrid motors are driven by the proton motive force.

The role of loop and β-turn residues as structural and functional determinants for the lipoyl domain from the Escherichia coli 2-oxoglutarate dehydrogenase complex

Biochemical Journal ◽

10.1042/bj20071119 ◽

2007 ◽

Vol 409 (2) ◽

pp. 357-366 ◽

Cited By ~ 12

Author(s):

D. Dafydd Jones ◽

Richard N. Perham

Keyword(s):

Escherichia Coli ◽

Structural Integrity ◽

Pyruvate Decarboxylase ◽

Covalent Attachment ◽

E Coli ◽

Loop Sequence ◽

Equivalent Residue ◽

Oxoglutarate Dehydrogenase ◽

And Function ◽

Lipoyl Domain

The lipoyl domain of the dihydrolipoyl succinyltransferase (E2o) component of the 2OGDH (2-oxoglutarate dehydrogenase) multienzyme complex houses the lipoic acid cofactor through covalent attachment to a specific lysine side chain residing at the tip of a β-turn. Residues within the lipoyl-lysine β-turn and a nearby prominent loop have been implicated as determinants of lipoyl domain structure and function. Protein engineering of the Escherichia coli E2o lipoyl domain (E2olip) revealed that removal of residues from the loop caused a major structural change in the protein, which rendered the domain incapable of reductive succinylation by 2-oxoglutarate decarboxylase (E1o) and reduced the lipoylation efficiency. Insertion of a new loop corresponding to that of the E. coli pyruvate dehydrogenase lipoyl domain (E2plip) restored lipoylation efficiency and the capacity to undergo reductive succinylation returned, albeit at a lower rate. Exchange of the E2olip loop sequence significantly improved the ability of the domain to be reductively acetylated by pyruvate decarboxylase (E1p), retaining approx. 10-fold more acetyl groups after 25 min than wild-type E2olip. Exchange of the β-turn residue on the N-terminal side of the E2o lipoyl-lysine DKA/V motif to the equivalent residue in E2plip (T42G), both singly and in conjunction with the loop exchange, reduced the ability of the domain to be reductively succinylated, but led to an increased capacity to be reductively acetylated by the non-cognate E1p. The T42G mutation also slightly enhanced the lipoylation rate of the domain. The surface loop is important to the structural integrity of the protein and together with Thr42 plays an important role in specifying the interaction of the lipoyl domain with its partner E1o in the E. coli 2OGDH complex.

Escherichia coli engineered to synthesize isopentenyl diphosphate and dimethylallyl diphosphate from mevalonate: a novel system for the genetic analysis of the 2-C-methyl-d-erythritol 4-phosphate pathway for isoprenoid biosynthesis

Biochemical Journal ◽

10.1042/bj3530059 ◽

2000 ◽

Vol 353 (1) ◽

pp. 59-67 ◽

Cited By ~ 47

Author(s):

Narciso CAMPOS ◽

Manuel RODRÍGUEZ-CONCEPCIÓN ◽

Susanna SAURET-GÜETO ◽

Francesca GALLEGO ◽

Luisa-María LOIS ◽

...

Keyword(s):

Escherichia Coli ◽

Genetic Analysis ◽

Mevalonate Pathway ◽

Mep Pathway ◽

Isopentenyl Diphosphate ◽

Isopentenyl Diphosphate Isomerase ◽

E Coli ◽

Dimethylallyl Diphosphate ◽

And Function ◽

Synthetic Operon

Isopentenyl diphosphate (IPP) and its isomer dimethylallyl diphosphate (DMAPP) constitute the basic building block of isoprenoids, a family of compounds that is extraordinarily diverse in structure and function. IPP and DMAPP can be synthesized by two independent pathways: the mevalonate pathway and the recently discovered 2-C-methyl-d-erythritol 4-phosphate (MEP) pathway. Although the MEP pathway is essential in most eubacteria, algae and plants and has enormous biotechnological interest, only some of its steps have been determined. We devised a system suitable for the genetic analysis of the MEP pathway in Escherichia coli. A synthetic operon coding for yeast 5-diphosphomevalonate decarboxylase, human 5-phosphomevalonate kinase, yeast mevalonate kinase and E. coli isopentenyl diphosphate isomerase was incorporated in the chromosome of this bacterium. The expression of this operon allowed the synthesis of IPP and DMAPP from mevalonate added exogenously and complementation of lethal mutants of the MEP pathway. We used this system to show that the ygbP, ychB and ygbB genes are essential in E. coli and that the steps catalysed by the products of these genes belong to the trunk line of the MEP pathway.

YbfA Regulates the Sensitivity of Escherichia coli K12 to Plantaricin BM-1 via the BasS/BasR Two-Component Regulatory System

Frontiers in Microbiology ◽

10.3389/fmicb.2021.659198 ◽

2021 ◽

Vol 12 ◽

Author(s):

Xinyue Chen ◽

Yifei Liu ◽

Junhua Jin ◽

Hui Liu ◽

Yanling Hao ◽

...

Keyword(s):

Escherichia Coli ◽

Cell Membrane ◽

Metabolic Pathways ◽

Gram Negative Bacteria ◽

Two Component System ◽

Proteomics Analysis ◽

Component System ◽

E Coli ◽

Two Component ◽

Plantaricin BM-1, a class IIa bacteriocin produced by Lactobacillus plantarum BM-1, shows obvious antibacterial activity against Escherichia coli. However, the mechanism underlying the action of class IIa bacteriocins against gram-negative bacteria remains to be explored. The purpose of this study was to investigate the role of YbfA, a DUF2517 domain-containing protein, in the response of Escherichia coli K12 to plantaricin BM-1. The growth curve experiment and MIC experiment showed that the sensitivity of E. coli to plantaricin BM-1 was decreased by a ybfA null mutation. Electron microscopy showed that the ybfA null mutation reduced the surface rupture and contraction caused by plantaricin BM-1, and mitigated the effect of plantaricin BM-1 on the morphology of the E. coli cell membrane. Proteomics analysis showed that 323 proteins were differentially expressed in E. coli lacking the ybfA gene (P < 0.05); 118 proteins were downregulated, and 205 proteins were upregulated. The metabolic pathways containing the upregulated proteins mainly included outer membrane proteins, integral components of the plasma membrane, regulation of cell motility, and regulation of locomotion. The metabolic pathways involving the downregulated proteins mainly included outer membrane protein glycine betaine transport, amino-acid betaine transport, and transmembrane signaling receptor activity. The results of the proteomics analysis showed that the protein expression of the BasS/BasR two-component system was significantly increased (P < 0.05). Moreover, the expression levels of downstream proteins regulated by this two-component system were also significantly increased, including DgkA, FliC, and MlaE, which are involved in cell membrane structure and function, and RT-qPCR also confirmed this result. The growth curve showed that the sensitivity of E. coli to plantaricin BM-1 was significantly increased due to deletion of the BasS/BasR two-component system. Thus, deletion of ybfA in E. coli can increase the expression of the BasS/BasR two-component system and positively regulate the structure and function of the cell membrane to reduce the sensitivity to plantaricin BM-1. This will help to explore the mechanism of action of class IIa bacteriocins against gram-negative bacteria.

A benchmark of protein solubility prediction methods on UDP-dependent glycosyltransferases

10.1101/2020.02.28.962894 ◽

2020 ◽

Author(s):

Fatemeh Ashari Ghomi ◽

Tiia Kittilä ◽

Ditte Hededam Welner

Keyword(s):

Escherichia Coli ◽

Protein Solubility ◽

Chemical Properties ◽

Prediction Methods ◽

Solubility Prediction ◽

E Coli ◽

Physico Chemical ◽

And Function ◽

Better Than

AbstractUDP-dependent glycosyltransferases (UGTs) are enzymes that glycosylate a wide variety of natural products, thereby modifying their physico-chemical properties, i.e. solubility, stability, reactivity, and function. To successfully leverage the UGTs in biocatalytic processes, we need to be able to screen and characterise them in vitro, which requires efficient heterologous expression in amenable hosts, preferably Escherichia coli. However, many UGTs are insoluble when expressed in standard and attempted optimised E. coli conditions, resulting in many unproductive and costly experiments. To overcome this limitation, we have investigated the performance of 11 existing solubility predictors on a dataset of 57 UGTs expressed in E. coli. We show that SoluProt outperforms other methods in terms of both threshold-independent and threshold-dependent measures. Among the benchmarked methods, only SoluProt is significantly better than random predictors using both measures. Moreover, we show that SoluProt uses a threshold for separating soluble and insoluble proteins that is optimal for our dataset. Hence, we conclude that using SoluProt to select UGT sequences for in vitro investigation will significantly increase the success rate of soluble expression, thereby minimising cost and enabling efficient characterisation efforts for biocatalysis research.