Screening of genes involved in phage-resistance of Escherichia coli and effects of substances interacting with primosomal protein A on the resistant bacteria.

AIMS: The study was to identify the genes involved in phage resistance and to develop an effective biocontrol method to improve the lytic activity of phages against foodborne pathogens. METHODS AND RESULTS: A total of 3,909 single gene-deletion mutants of Escherichia coli BW25113 from the Keio collection were individually screened for genes involved in phage resistance. Phage S127BCL3 isolated from chicken liver, infecting both E. coli BW25113 and O157: H7, was characterized and used for screening. The 10 gene-deletion mutants showed increased susceptibility to phage S127BCL3. Among them, priA gene-deletion mutant strain showed significant susceptibility to the phages S127BCL3 and T7. Furthermore, we investigated the substances that have been reported to inhibit the function of primosomal protein A (PriA) and were used to confirm increased phage susceptibility in E. coli BW25113 (Parent strain) and O157: H7. CONCLUSION: PriA inhibitors at a low concentration showed combined effects with phage against E. coli O157: H7 and delayed the regrowth rate of phage-resistant cells.

The Bacillus subtilis PriA Winged Helix Domain Is Critical for Surviving DNA Damage.

DNA replication forks regularly encounter lesions or other impediments that result in a blockage to fork progression. PriA is one of the key proteins used by virtually all eubacteria to survive conditions that result in a blockage to replication fork movement. PriA directly binds stalled replication forks and initiates fork restart allowing for chromosomes to be fully duplicated under stressful conditions. We used a CRISPR-Cas gene editing approach to map PriA residues critical for surviving DNA damage induced by several antibiotics in B. subtilis. We find that the winged helix (WH) domain in B. subtilis PriA is critical for surviving DNA damage and participates in DNA binding. The important in vivo function of the WH domain mapped to distinct surfaces that were also conserved among several Gram-positive human pathogens. In addition, we identified an amino acid linker neighboring the WH domain that is greatly extended in B. subtilis due to an insertion. Shortening this linker induced a hypersensitive phenotype to DNA damage, suggesting that its extended length is critical for efficient replication fork restart in vivo. Because the WH domain is dispensable in E. coli PriA, our findings demonstrate an important difference in the contribution of the WH domain during fork restart in B. subtilis. Furthermore, with our results we suggest that this highly variable region in PriA could provide different functions across diverse bacterial organisms. IMPORTANCE PriA is an important protein found in virtually all bacteria that recognizes stalled replication forks orchestrating fork restart. PriA homologs contain a winged helix (WH) domain. The E. coli PriA WH domain is dispensable and functions in a fork restart pathway that is not conserved outside of E. coli and closely related proteobacteria. We analyzed the importance of the WH domain and an associated linker in B. subtilis and found that both are critical for surviving DNA damage. This function mapped to a small motif at the C-terminal end of the WH domain, which is also conserved in pathogenic bacteria. The motif was not required for DNA binding and therefore may perform a novel function in the replication fork restart pathway.

A Complexed Crystal Structure of a Single-Stranded DNA-Binding Protein with Quercetin and the Structural Basis of Flavonol Inhibition Specificity.

Single-stranded DNA (ssDNA)-binding protein (SSB) plays a crucial role in DNA replication, repair, and recombination as well as replication fork restarts. SSB is essential for cell survival and, thus, is an attractive target for potential antipathogen chemotherapy. Whether naturally occurring products can inhibit SSB remains unknown. In this study, the effect of the flavonols myricetin, quercetin, kaempferol, and galangin on the inhibition of Pseudomonas aeruginosa SSB (PaSSB) was investigated. Furthermore, SSB was identified as a novel quercetin-binding protein. Through an electrophoretic mobility shift analysis, myricetin could inhibit the ssDNA binding activity of PaSSB with an IC50 of 2.8 ± 0.4 µM. The effect of quercetin, kaempferol, and galangin was insignificant. To elucidate the flavonol inhibition specificity, the crystal structure of PaSSB complexed with the non-inhibitor quercetin was solved using the molecular replacement method at a resolution of 2.3 Å (PDB entry 7VUM) and compared with a structure with the inhibitor myricetin (PDB entry 5YUN). Although myricetin and quercetin bound PaSSB at a similar site, their binding poses were different. Compared with myricetin, the aromatic ring of quercetin shifted by a distance of 4.9 Å and an angle of 31o for hydrogen bonding to the side chain of Asn108 in PaSSB. In addition, myricetin occupied and interacted with the ssDNA binding sites Lys7 and Glu80 in PaSSB whereas quercetin did not. This result might explain why myricetin could, but quercetin could not, strongly inhibit PaSSB. This molecular evidence reveals the flavonol inhibition specificity and also extends the interactomes of the natural anticancer products myricetin and quercetin to include the OB-fold protein SSB.

Characterization of the Chimeric PriB-SSBc Protein.

PriB is a primosomal protein required for the replication fork restart in bacteria. Although PriB shares structural similarity with SSB, they bind ssDNA differently. SSB consists of an N-terminal ssDNA-binding/oligomerization domain (SSBn) and a flexible C-terminal protein-protein interaction domain (SSBc). Apparently, the largest difference in structure between PriB and SSB is the lack of SSBc in PriB. In this study, we produced the chimeric PriB-SSBc protein in which Klebsiella pneumoniae PriB (KpPriB) was fused with SSBc of K. pneumoniae SSB (KpSSB) to characterize the possible SSBc effects on PriB function. The crystal structure of KpSSB was solved at a resolution of 2.3 Å (PDB entry 7F2N) and revealed a novel 114-GGRQ-117 motif in SSBc that pre-occupies and interacts with the ssDNA-binding sites (Asn14, Lys74, and Gln77) in SSBn. As compared with the ssDNA-binding properties of KpPriB, KpSSB, and PriB-SSBc, we observed that SSBc could significantly enhance the ssDNA-binding affinity of PriB, change the binding behavior, and further stimulate the PriA activity (an initiator protein in the pre-primosomal step of DNA replication), but not the oligomerization state, of PriB. Based on these experimental results, we discuss reasons why the properties of PriB can be retrofitted when fusing with SSBc.

Function of a strand-separation pin element in the PriA DNA replication restart helicase.

DNA helicases are motor proteins that couple the chemical energy of nucleoside triphosphate hydrolysis to the mechanical functions required for DNA unwinding. Studies of several helicases have identified strand-separating "pin" structures that are positioned to intercept incoming dsDNA and promote strand separation during helicase translocation. However, pin structures vary among helicases and it remains unclear whether they confer a conserved unwinding mechanism. Here, we tested the biochemical and cellular roles of a putative pin element within the Escherichia coli PriA DNA helicase. PriA orchestrates replication restart in bacteria by unwinding the lagging-strand arm of abandoned DNA replication forks and reloading the replicative helicase with the help of protein partners that combine with PriA to form what is referred to as a primosome complex. Using in vitro protein-DNA cross-linking, we localized the putative pin (a ß-hairpin within a zinc-binding domain in PriA) near the ssDNA-dsDNA junction of the lagging strand in a PriA-DNA replication fork complex. Removal of residues at the tip of the ß-hairpin eliminated PriA DNA unwinding, interaction with the primosome protein PriB, and cellular function. We isolated a spontaneous intragenic suppressor mutant of the priA ß-hairpin deletion mutant in which 22 codons around the deletion site were duplicated. This suppressor variant and an Ala-substituted ß-hairpin PriA variant displayed wildtype levels of DNA unwinding and PriB binding in vitro These results suggest essential but sequence nonspecific roles for the PriA pin element and coupling of PriA DNA unwinding to its interaction with PriB.

Co-occurrence of analogous enzymes determines evolution of a novel (ßα)8-isomerase sub-family after non-conserved mutations in flexible loop.

We investigate the evolution of co-occurring analogous enzymes involved in L-tryptophan and L-histidine biosynthesis in Actinobacteria Phylogenetic analysis of trpF homologues, a missing gene in certain clades of this lineage whose absence is complemented by a dual-substrate HisA homologue, termed PriA, found that they fall into three categories: (i) trpF-1, an L-tryptophan biosynthetic gene horizontally acquired by certain Corynebacterium species; (ii) trpF-2, a paralogue known to be involved in synthesizing a pyrrolopyrrole moiety and (iii) trpF-3, a variable non-conserved orthologue of trpF-1 We previously investigated the effect of trpF-1 upon the evolution of PriA substrate specificity, but nothing is known about the relationship between trpF-3 and priA After in vitro steady-state enzyme kinetics we found that trpF-3 encodes a phosphoribosyl anthranilate isomerase. However, mutation of this gene in Streptomyces sviceus did not lead to auxothrophy, as expected from the biosynthetic role of trpF-1 Biochemical characterization of a dozen co-occurring TrpF-2 or TrpF-3, with PriA homologues, explained the prototrophic phenotype, and unveiled an enzyme activity trade-off between TrpF and PriA. X-ray structural analysis suggests that the function of these PriA homologues is mediated by non-conserved mutations in the flexible L5 loop, which may be responsible for different substrate affinities. Thus, the PriA homologues that co-occur with TrpF-3 represent a novel enzyme family, termed PriB, which evolved in response to PRA isomerase activity. The characterization of co-occurring enzymes provides insights into the influence of functional redundancy on the evolution of enzyme function, which could be useful for enzyme functional annotation.

Identification of Subunit Binding Positions on a Model Fork and Displacements That Occur during Sequential Assembly of the Escherichia coli Primosome.

When replication stalls and forks disassemble, the restart primosome is required to reload the replicative helicase so that chromosomal replication can be reinitiated. We have taken a photo-cross-linking approach, using model replication forks containing a phenyl diazirine placed at single locations, to determine the positions of primosomal protein binding and changes in interactions that occur during the assembly reaction. This approach revealed a novel mode for single-stranded DNA-binding protein (SSB)-DNA binding, in which SSB interacts with both the leading and lagging single-strand segments and the parental duplex of the fork. Cross-linking to a novel region within SSB is observed only when it is bound to forked structures. This binding mode is also followed by PriB. PriA binds to the fork, excluding SSB and PriB, interacting with the primer terminus, single-stranded leading and lagging strands and duplex in immediate proximity of the fork. SSB binds to flanking single-stranded segments distal to the fork in the presence of PriA. The addition of PriB or DnaT to a PriA-SSB-fork complex does not lead to cross-linking or displacement, suggesting that their association is through protein-protein interactions at early stages of the reaction. Upon addition of DnaC and the DnaB helicase in the presence of ATPγS, helicase is assembled, leading to contacts within the duplex region on the tracking (lagging) strand and strong contacts with the displaced leading single strand near the fork. PriA is displaced from DNA upon helicase assembly.

Fork restart protein, PriA, binds around oriC after depletion of nucleotide precursors: Replication fork arrest near the replication origin.

Arrest of replication fork progression is one of the most common causes for increasing the genomic instability. In bacteria, PriA, a conserved DEXH-type helicase, plays a major role in recognition of the stalled forks and restart of DNA replication. We took advantage of PriA's ability to specifically recognize stalled replication forks to determine the genomic loci where replication forks are prone to stall on the Escherichia coli genome. We found that PriA binds around oriC upon thymine starvation which reduces the nucleotide supply and causes replication fork stalling. PriA binding quickly disappeared upon readdition of thymine. Furthermore, BrdU was incorporated at around oriC upon release from thymine starvation. Our results indicate that reduced supply of DNA replication precursors causes replication fork stalling preferentially in the 600 kb segment centered at oriC. This suggests that replication of the vicinity of oriC requires higher level of nucleotide precursors. The results also point to a possibility of slow fork movement and/or the presence of multiple fork arrest signals within this segment. Indeed, we have identified rather strong fork stall/pausing signals symmetrically located at ∼50 kb away from oriC. We speculate that replication pausing and fork-slow-down shortly after initiation may represent a novel checkpoint that ensures the presence of sufficient nucleotide supply prior to commitment to duplication of the entire genome.

Structural, kinetic, and evolutionary peculiarities of HISN3, a plant 5'-ProFAR isomerase.

Histidine biosynthesis is essential for the growth and development of plants, where it occurs within chloroplasts. The eleven reactions are catalyzed by eight enzymes, known as HISN1-8, each acting sequentially. Here, we present the crystal structures of a 5'-ProFAR isomerase (HISN3) from the model legume Medicago truncatula bound to its enzymatically synthesized substrate (ProFAR) and product (PrFAR). The active site of MtHISN3 contains a sodium cation that participates in ligand recognition, a feature not observed in bacterial and fungal structures of homologous enzymes. The steady-state kinetics of wild-type MtHISN3 revealed a slightly higher turnover rate compared to its bacterial homologs. Plant HISN3 sequences contain an unusually elongated Lys60-Ser91 fragment, while deletion of the 74-80 region resulted in a 30-fold loss in catalytic efficiency compared to the wild-type. Molecular dynamics simulations suggested that the fragment facilitates product release, thereby contributing to a higher kcat. Moreover, conservation analyses suggested a non-cyanobacterial origin for plant HISN3 enzymes, which is another instance of a non-cyanobacterial enzyme in the plant histidine biosynthetic pathway. Finally, a virtual screening campaign yielded five molecules, with the energy gains ranging between -13.6 and -13.1 kcal/mol, which provide new scaffolds for the future development of herbicides.

Identification of genetic interactions with priB links the PriA/PriB DNA replication restart pathway to double-strand DNA break repair in Escherichia coli.

Collisions between DNA replication complexes (replisomes) and impediments such as damaged DNA or proteins tightly bound to the chromosome lead to premature dissociation of replisomes at least once per cell cycle in Escherichia coli. Left unrepaired, these events produce incompletely replicated chromosomes that cannot be properly partitioned into daughter cells. DNA replication restart, the process that reloads replisomes at prematurely terminated sites, is therefore essential in E. coli and other bacteria. Three replication restart pathways have been identified in E. coli: PriA/PriB, PriA/PriC, and PriC/Rep. A limited number of genetic interactions between replication restart and other genome maintenance pathways have been defined, but a systematic study placing replication restart reactions in a broader cellular context has not been performed. We have utilized transposon-insertion sequencing to identify new genetic interactions between DNA replication restart pathways and other cellular systems. Known genetic interactors with the priB replication restart gene (uniquely involved in the PriA/PriB pathway) were confirmed and several novel priB interactions were discovered. Targeted genetic and imaging-based experiments with priB and its genetic partners revealed significant double-strand DNA break accumulation in strains with mutations in dam, rep, rdgC, lexA, or polA. Modulating the activity of the RecA recombinase partially suppressed the detrimental effects of rdgC or lexA mutations in ΔpriB cells. Taken together, our results highlight roles for several genes in double-strand DNA break homeostasis and define a genetic network that facilitates DNA repair/processing upstream of PriA/PriB-mediated DNA replication restart in E. coli.

TT-pocket/HIRAN: binding to 3'-terminus of DNA for recognition and processing of stalled replication forks.

Stalled replication forks need to be swiftly detected and protected from collapse and the cause for fork stall be removed to restore the active replication fork. In bacteria, stalled forks are recognized and stabilized by PriA, a DEXH-type helicase, which also facilitates reassembly of an active replication fork. A TT-pocket (three-prime terminus binding pocket) present in the N-terminal segment of PriA plays a crucial role in stabilization of the stalled forks by specifically binding to the 3$^\prime$-terminus of the nascent leading strand. Eukaryotic proteins, Rad5/HLTF, contain a TT-pocket related domain, HIRAN, that specifically binds to 3'-terminus of DNA and play a role in stalled fork processing. While the TT-pocket of PriA facilitates the formation of an apparently stable and immobile complex on a fork with a 3'-terminus at the fork junction, HIRAN of Rad5/HLTF facilitates fork regression by itself. A recent report shows that HIRAN can displace 3 nucleotides at the end of the duplex DNA, providing mechanistic insight into how stalled forks are reversed in eukaryotes. In this article, I will compare the roles of 3'-terminus binding domains in stalled fork processing in prokaryotes and in eukaryotes.

Use of an unnatural amino acid to map helicase/DNA interfaces via photoactivated crosslinking.

Formation of protein/nucleic acid complexes is essential for life. From DNA replication and repair to transcription and translation, myriad different proteins bind nucleic acids to execute their essential cellular functions. Our understanding of the mechanisms underlying recognition and processing of nucleic acids can be greatly informed by mapping protein domains and residues that form interfaces with their DNA or RNA targets. Here we describe a crosslinking protocol in which the unnatural amino acid p-benzoyl-l-phenylalanine (Bpa) integrated at selected sites within the PriA DNA helicase is used to map surfaces of the protein that interact with specific positions in a synthetic DNA replication fork in vitro.

Comparing SSB-PriA Functional and Physical Interactions in Gram-Positive and -Negative Bacteria.

Single-stranded DNA (ssDNA)-binding protein (SSB) is essential for DNA metabolic processes. SSB also binds to many DNA-binding proteins that constitute the SSB interactome. The mechanism through which PriA helicase, an initiator protein in the DNA replication restart process, is stimulated by SSB in Escherichia coli (EcSSB) has been established. However, some Gram-positive bacterial SSBs such as Bacillus subtilis SsbA (a counterpart of EcSSB), Staphylococcus aureus SsbA, SsbB, and SsbC do not activate PriA helicase. Here, we describe some of the methods used in our laboratory to compare SSB-PriA functional and physical interactions in Gram-positive and -negative bacteria.

In Vivo Binding of Single-Stranded DNA-Binding Protein to Stalled Replication Fork Helicases.

Understanding protein-protein interactions is key to unraveling protein function in vivo. Here we describe a dual/triple-plasmid system that enables co-expression of two, or three, recombinant proteins harboring different affinity tags in the same Escherichia coli cell. This novel protein expression system provides a platform to understand protein-protein interactions and enables researchers to study protein complex formation and in vivo localization.

Characterize the Interaction of the DNA Helicase PriA with the Stalled DNA Replication Fork Using Atomic Force Microscopy.

In bacteria, the restart of stalled DNA replication forks requires the DNA helicase PriA. PriA can recognize and remodel abandoned DNA replication forks, unwind DNA in the 3'-to-5' direction, and facilitate the loading of the helicase DnaB onto the DNA to restart replication. ssDNA-binding protein (SSB) is typically present at the abandoned forks, protecting the ssDNA from nucleases. Research that is based on the assays for junction dissociation, surface plasmon resonance, single-molecule FRET, and x-ray crystal structure has revealed the helicase activity of PriA, the SSB-PriA interaction, and structural information of PriA helicase. Here, we used Atomic Force Microscopy (AFM) to visualize the interaction between PriA and DNA substrates with or without SSB in the absence of ATP to delineate the substrate recognition pattern of PriA before its ATP-catalyzed DNA-unwinding reaction. The protocol describes the steps to obtain high-resolution AFM images and the details of data analysis and presentation.

Novel thermostable lipase produced by a thermo-halophilic bacterium that catalyses hydrolytic and transesterification reactions.

Lipase belongs to the class of hydrolytic enzymes that are widely used in the biotechnology industries. The goal of this research was to purify and characterize lipase produced from a thermo-halophilic bacterium, namely Pria Laot Sabang 80 (PLS 80). Purification was performed using ammonium sulphate fractionation, followed by gel filtration chromatography using Sepharose Cl-6B. After purification, the enzyme had a specific activity of 326.6 U/mg with a purity of 6.02 higher than the crude extract; with a molecular weight of around 50 kDa. The optimum activity was observed at 70 °C and pH 9. The activity increased in the presence of 10 mM Mn2+, K+ and Ca2+ ions, while Hg2+ only slightly increased the enzyme activity. In contrast, the activity decreased in 10 mM Mg2+, Zn2+, Co2+, EDTA, and PMSF. The enzyme showed good hydrolytic activity on long fatty acids substrates (p-nitrophenyl palmitate) with a value of 35.5 U/mL. It was also able to catalyze a transesterification reaction. GC-MS result showed that the biodiesel consisted of methyl octanoate (5.3%), methyl caprate (12.4%), methyl laurate (34.1%), methyl myristate (10.7%), methyl palmitate (3.9%), and methyl stearate (1.2%) when using coconut oil as the substrate. The results suggested that the lipase from PLS 80 had unique attributes that could be useful in various industrial applications.

Supercoiling, R-loops, Replication and the Functions of Bacterial Type 1A Topoisomerases.

Type 1A topoisomerases (topos) are the only topos that bind single-stranded DNA and the only ones found in all cells of the three domains of life. Two subfamilies, topo I and topo III, are present in bacteria. Topo I, found in all of them, relaxes negative supercoiling, while topo III acts as a decatenase in replication. However, recent results suggest that they can also act as back-up for each other. Because they are ubiquitous, type 1A enzymes are expected to be essential for cell viability. Single topA (topo I) and topB (topo III) null mutants of Escherichia coli are viable, but for topA only with compensatory mutations. Double topA topB null mutants were initially believed to be non-viable. However, in two independent studies, results of next generation sequencing (NGS) have recently shown that double topA topB null mutants of Bacillus subtilis and E. coli are viable when they carry parC parE gene amplifications. These genes encode the two subunits of topo IV, the main cellular decatenase. Here, we discuss the essential functions of bacterial type 1A topos in the context of this observation and new results showing their involvement in preventing unregulated replication from R-loops.

SAAV2152 is a single-stranded DNA binding protein: the third SSB in Staphylococcus aureus.

Single-stranded DNA-binding proteins (SSBs) play crucial roles in DNA replication, repair, and recombination. Unlike E. coli, which contains only one type of SSB (EcSSB), some bacteria have two paralogous SSBs, namely, SsbA and SsbB. In this study, we found the third SSB-like protein in Staphylococcus aureus, SAAV2152, which was designated as SaSsbC. SaSsbC is a protein of 131 amino acids and shares 38%, 36%, and 33% sequence identity to SaSsbB, SaSsbA, and EcSSB, respectively. Gene map analysis showed that unlike the E. coli ssb gene, which is adjacent to uvrA gene, the S. aureus ssb gene SAAV2152 is flanked by the putative SceD, the putative YwpF, and fabZ genes. A homology model showed that SaSsbC consists of the classic oligonucleotide/oligosaccharide-binding fold at the N-terminus. At the C-terminus, SaSsbC did not exhibit sequence similarity to that of EcSSB. Electrophoretic mobility shift analysis showed that SaSsbC formed a single complex with ssDNA of different lengths. Mutational analysis revealed that Tyr36, Tyr47, Phe53, and Tyr81 in SaSsbC are at positions that structurally correspond to the important residues of EcSSB for binding to ssDNA and are also critical for SaSsbC to bind ssDNA. Unlike EcSSB, which can stimulate EcPriA, SaSsbC did not affect the activity of SaPriA. In addition, SaSsbA inhibitor 9-methyl-2,3,7-trihydroxy-6-fluorone (NSC5426) could inhibit the ssDNA-binding activity of SaSsbC with IC50 of 78 µM. In conclusion, this study has identified and characterized SAAV2152 as a kind of SSB, and further research can directly focus on determining its actual physiological role in S. aureus.

Replication Fork Breakage and Restart in Escherichia coli.

In all organisms, replication impairments are an important source of genome rearrangements, mainly because of the formation of double-stranded DNA (dsDNA) ends at inactivated replication forks. Three reactions for the formation of dsDNA ends at replication forks were originally described for Escherichia coli and became seminal models for all organisms: the encounter of replication forks with preexisting single-stranded DNA (ssDNA) interruptions, replication fork reversal, and head-to-tail collisions of successive replication rounds. Here, we first review the experimental evidence that now allows us to know when, where, and how these three different reactions occur in E. coli. Next, we recall our recent studies showing that in wild-type E. coli, spontaneous replication fork breakage occurs in 18% of cells at each generation. We propose that it results from the replication of preexisting nicks or gaps, since it does not involve replication fork reversal or head-to-tail fork collisions. In the recB mutant, deficient for double-strand break (DSB) repair, fork breakage triggers DSBs in the chromosome terminus during cell division, a reaction that is heritable for several generations. Finally, we recapitulate several observations suggesting that restart from intact inactivated replication forks and restart from recombination intermediates require different sets of enzymatic activities. The finding that 18% of cells suffer replication fork breakage suggests that DNA remains intact at most inactivated forks. Similarly, only 18% of cells need the helicase loader for replication restart, which leads us to speculate that the replicative helicase remains on DNA at intact inactivated replication forks and is reactivated by the replication restart proteins.

Enzyme evolution beyond gene duplication: A model for incorporating horizontal gene transfer.

Understanding the evolution of enzyme function after gene duplication has been a major goal of molecular biologists, biochemists and evolutionary biologists alike, for almost half a century. In contrast, the impact that horizontal gene transfer (HGT) has had on the evolution of enzyme specialization and the assembly of metabolic networks has just started to being investigated. Traditionally, evolutionary studies of enzymes have been limited to either the function of enzymes in vitro, or to sequence variability at the population level, where in almost all cases the starting conceptual framework embraces gene duplication as the mechanism responsible for the appearance of genetic redundancy. Very recently, we merged comparative phylogenomics, detection of selection signals, enzyme kinetics, X-ray crystallography and computational molecular dynamics, to characterize the sub-functionalization process of an amino acid biosynthetic enzyme prompted by an episode of HGT in bacteria. Some of the evolutionary implications of these functional studies, including a proposed model of enzyme specialization independent of gene duplication, are developed in this commentary.