Geobacillus Kaustophilus HTA426 Research Articles

A novel alkane monooxygenase evolved from a broken piece of ribonucleotide reductase in Geobacillus kaustophilus HTA426 isolated from Mariana Trench

We have accidentally found that a thermophilic Geobacillus kaustophilus HTA426 is capable of degrading alkanes although it has no alkane oxygenating enzyme genes. Our experimental results revealed that a putative ribonucleotide reductase small subunit GkR2loxI (GK2771) gene encodes a novel heterodinuclear Mn–Fe alkane monooxygenase/hydroxylase. GkR2loxI protein can perform two-electron oxidations similar to homonuclear diiron bacterial multicomponent soluble methane monooxygenases. This finding not only answers a long-standing question about the substrate of the R2lox protein clade, but also expands our understanding of the vast diversity and new evolutionary lineage of the bacterial alkane monooxygenase/hydroxylase family.

Genes encoding a novel thermostable bacteriocin in the thermophilic bacterium Aeribacillus pallidus PI8.

Aeribacillus pallidus PI8 is a gram-positive thermophilic bacterium that produces thermostable antimicrobial substances against several bacterial species, including Geobacillus kaustophilus HTA426. In the present study, we sought to identify genes of PI8 with antibacterial activity. We isolated, cloned, and characterized a thermostable bacteriocin from A. pallidus PI8 and named it pallidocyclin. Mass spectrometric analyses of pallidocyclin revealed that it had a circular peptide structure, and its precursor was encoded by pcynA in the PI8 genome. pcynA is the second gene within the pcynBACDEF operon. Expression of the full-length pcynBACDEF operon in Bacillus subtilis produced intact pallidocyclin, whereas expression of pcynF in G. kaustophilus HTA426 conferred resistance to pallidocyclin. A. pallidus PI8 possesses the pcynBACDEF operon to produce pallidocyclin. pcynA encodes the pallidocyclin precursor, and pcynF acts as an antagonist of pallidocyclin.

Development of a thermophilic host-vector system for the production of recombinant proteins at elevated temperatures.

Geobacillus spp. are moderate thermophiles that can efficiently produce recombinant proteins. Considering the protein production exhibited by these species, we searched for robust promoters in Geobacillus kaustophilus HTA426. Transcriptome data revealed that several genes were highly expressed during the proliferative phase; their promoters were characterized using reporter assays with Venus fluorescent protein (VFP). The results suggested that the cspD promoter (PcspD) directed robust vfp expression at 60°C in G. kaustophilus. Although cspD potentially encodes a cold-shock protein, PcspD functioned at elevated temperatures. The promoter strongly functioned even in Escherichia coli; this prevented the cloning of some genes (e.g., vfp) downstream of it on a plasmid vector via E. coli-based genetic manipulation. Consequently, we generated a mutated PcspD that functioned inefficiently in E. coli and constructed the pGKE124 plasmid using the mutant promoter. The plasmid could carry vfp in E. coli and afford the production of VFP in G. kaustophilus at a yield of 390 mg/L. pGKE124 directed a similar production in other thermophilic species; the highest yield was observed in Geobacillus thermodenitrificans K1041. Several proteins could be produced using a system involving G. thermodenitrificans K1041 and pGKE124. Notably, the extracellular production of xylanase at a yield of 1 g/L was achieved using this system. Although the leaky production of nonsecretory proteins was observed, we developed a simple process to collectively purify recombinant proteins from the intracellular and extracellular fractions. The findings presented there propose an effective host-vector system for the production of recombinant proteins at elevated temperatures. KEY POINTS: • A thermophilic system to produce recombinant proteins was constructed. • The system produced diverse proteins using inexpensive media at elevated temperatures. • The system produced an extracellular protein at a yield of 1 g/L of culture.

Frequent Transposition of Multiple Insertion Sequences in Geobacillus kaustophilus HTA426.

Geobacillus kaustophilus HTA426 is a thermophilic bacterium whose genome harbors numerous insertion sequences (IS). This study was initially conducted to generate mutant genes for thermostable T7 RNA polymerase in G. kaustophilus; however, relevant experiments unexpectedly identified that the organism transposed multiple IS elements and produced derivative cells that expressed a silent gene via transposition. The transposed elements were diverse and included members of the IS4, IS701, IS1634, and ISLre2 families. The transposition was relatively active at elevated temperatures and generated 4–9 bp of direct repeats at insertion sites. Transposition was more frequent in proliferative cells than in stationary cells but was comparable between both cells when sigX, which encodes an extra-cytoplasmic function sigma factor, was forcibly expressed. Southern blot analysis indicated that IS transposition occurred under growth inhibitory conditions by diverse stressors; however, IS transposition was not detected in cells that were cultured under growth non-inhibitory conditions. These observations suggest that G. kaustophilus enhances IS transposition via sigX-dependent stress responses when proliferative cells were prevented from active propagation. Considering Geobacillus spp. are highly adaptive bacteria that are remarkably distributed in diverse niches, it is possible that these organisms employ IS transposition for environmental adaptation via genetic diversification. Thus, this study provides new insights into adaptation strategies of Geobacillus spp. along with implications for strong codependence between mobile genetic elements and highly adaptive bacteria for stable persistence and evolutionary diversification, respectively. This is also the first report to reveal active IS elements at elevated temperatures in thermophiles and to suggest a sigma factor that governs IS transposition.

Identification of a repressor for the two iol operons required for inositol catabolism in Geobacillus kaustophilus.

Geobacillus kaustophilus HTA426, a thermophilic Gram-positive bacterium, feeds on inositol as its sole carbon source, and an iol gene cluster required for inositol catabolism has been postulated with reference to the iol genes in Bacillus subtilis. The iol gene cluster of G. kaustophilus comprises two tandem operons induced in the presence of inositol; however, the mechanism underlying this induction remains unclear. B. subtilis iolQ is known to be involved in the regulation of iolX encoding scyllo-inositol dehydrogenase, and its homologue in HTA426 was found two genes upstream of the first gene (gk1899) of the iol gene cluster and was termed iolQ in G. kaustophilus. When iolQ was inactivated in G. kaustophilus, not only cellular myo-inositol dehydrogenase activity due to gk1899 expression but also the transcription of the two iol operons became constitutive. IolQ was produced and purified as a C-terminal histidine (His)-tagged fusion protein in Escherichia coli and subjected to an in vitro gel electrophoresis mobility shift assay to examine its DNA-binding property. It was observed that IolQ bound to the DNA fragments containing each of the two iol promoter regions and that DNA binding was antagonized by myo-inositol. Moreover, DNase I footprinting analyses identified two tandem binding sites of IolQ within each of the iol promoter regions. By comparing the sequences of the binding sites, a consensus sequence for IolQ binding was deduced to form a palindrome of 5'-RGWAAGCGCTTSCY-3' (where R=A or G, W=A or T, S=G or C, and Y=C or T). IolQ functions as a transcriptional repressor regulating the induction of the two iol operons responding to myo-inositol.

A plasmid vector that directs hyperproduction of recombinant proteins in the thermophiles Geobacillus species.

Geobacillus spp. are moderate thermophiles that have great potential for use in diverse applications. For effective utilization of the species, genetic tools have been extensively studied; however, an overexpression vector remains to be developed. Here we constructed a plasmid vector that can shuttle between Escherichia coli and Geobacillus spp., and which contained a maltose-inducible promoter from Geobacillus kaustophilus HTA426. Although the vector (termed pGKE119) was originally designed for basal gene expression, it surprisingly directed robust protein production in G. kaustophilus. Protein production essentially occurred in an auto-inducible manner without maltose; however, some proteins were produced more efficiently in the presence of maltose. Although the productivity was affected by culture conditions, three proteins were successfully produced with abundance ratios of 12-27% (on a total protein basis) and yields of 77-170mg (per L culture). pGKE119 directed substantial protein production even in Geobacillus subterraneus, Geobacillus thermoglucosidasius, and Geobacillus thermoleovorans. This suggests that pGKE119 can use a range of Geobacillus spp. as hosts and widely expand their genetic toolbox. Because Geobacillus spp. are highly proliferative bacteria that are distinct from organisms used as protein production hosts, pGKE119 may also provide a novel platform for hyperproduction of recombinant proteins.

Antibiotic resistance mutations induced in growing cells of Bacillus-related thermophiles.

Stress-induced mutagenesis can assist pathogens in generating drug-resistant cells during antibiotic therapy; however, if and how antibiotics induce mutagenesis in microbes remains poorly understood. A non-pathogenic thermophile, Geobacillus kaustophilus HTA426, efficiently produces derivative cells resistant to rifampicin and streptomycin via rpoB and rpsL mutations, respectively. Here, we examined this phenomenon to suggest a novel mutagenic mode induced by antibiotics. Fluctuation analysis indicated that mutations occurred via spontaneous mutations during culture. However, mutations were much more frequent in growing cells than stationary cells, and mutation sites were varied through cell growth. These observations suggested that growing cells induced mutagenesis in response to antibiotics. An in-frame deletion of mfd, which governs transcription-coupled repair to correct DNA lesions on the transcribed strand, caused mutations that were comparable between growing and stationary cells; therefore, the mutagenic mechanism was attributable to DNA repair defects where growing cells depressed mfd function. Mutations occurred more frequently at optimal growth temperatures for G. kaustophilus than at a higher growth temperature, suggesting that the mutagenesis relies on active cellular activities rather than high temperature-associated DNA damage. In addition, the mutagenesis may involve a mutagenic factor targeting these sites, in addition to mfd depression, because rpoB and rpsL mutations were dominant at thymine and guanine sites on the transcribed strand. A similar mutagenic profile was observed for other Geobacillus and thermophilic Bacillus species. This suggests that Bacillus-related thermophiles commonly induce mutagenesis in response to rifampicin and streptomycin to produce resistant cells.

EM structure of a helicase-loader complex depicting a 6:2 binding sub-stoichiometry from Geobacillus kaustophilus HTA426

During DNA replication, bacterial helicase is recruited as a complex in association with loader proteins to unwind the parental duplex. Previous structural studies have reported saturated 6:6 helicase-loader complexes with different conformations. However, structural information on the sub-stoichiometric conformations of these previously-documented helicase-loader complexes remains elusive. Here, with the aid of single particle electron-microscopy (EM) image reconstruction, we present the Geobacillus kaustophilus HTA426 helicase-loader (DnaC-DnaI) complex with a 6:2 binding stoichiometry in the presence of ATPγS. In the 19 Å resolution EM map, the undistorted and unopened helicase ring holds a robust loader density above the C-terminal RecA-like domain. Meanwhile, the path of the central DNA binding channel appears to be obstructed by the reconstructed loader density, implying its potential role as a checkpoint conformation to prevent the loading of immature complex onto DNA. Our data also reveals that the bound nucleotides and the consequently induced conformational changes in the helicase hexamer are essential for active association with loader proteins. These observations provide fundamental insights into the formation of the helicase-loader complex in bacteria that regulates the DNA replication process.

Conjugative plasmid transfer from Escherichia coli is a versatile approach for genetic transformation of thermophilic Bacillus and Geobacillus species.

We previously demonstrated efficient transformation of the thermophile Geobacillus kaustophilus HTA426 using conjugative plasmid transfer from Escherichia coli BR408. To evaluate the versatility of this approach to thermophile transformation, this study examined genetic transformation of various thermophilic Bacillus and Geobacillus spp. using conjugative plasmid transfer from E. coli strains. E. coli BR408 successfully transferred the E. coli-Geobacillus shuttle plasmid pUCG18T to 16 of 18 thermophiles with transformation efficiencies between 4.1×10(-7) and 3.8×10(-2)/recipient. Other E. coli strains that are different from E. coli BR408 in intracellular DNA methylation also generated transformants from 9 to 15 of the 18 thermophiles, including one that E. coli BR408 could not transform, although the transformation efficiencies of these strains were generally lower than those of E. coli BR408. The conjugation was performed by simple incubation of an E. coli donor and a thermophile recipient without optimization of experimental conditions. Moreover, thermophile transformants were distinguished from abundant E. coli donor only by high temperature incubation. These observations suggest that conjugative plasmid transfer, particularly using E. coli BR408, is a facile and versatile approach for plasmid introduction into thermophilic Bacillus and Geobacillus spp., and potentially a variety of other thermophiles.

A thiostrepton resistance gene and its mutants serve as selectable markers in Geobacillus kaustophilus HTA426.

Effective utilization of microbes often requires complex genetic modification using multiple antibiotic resistance markers. Because a few markers have been used in Geobacillus spp., the present study was designed to identify a new marker for these thermophiles. We explored antibiotic resistance genes functional in Geobacillus kaustophilus HTA426 and identified a thiostrepton resistance gene (tsr) effective at 50°C. The tsr gene was further used to generate the mutant tsr(H258Y) functional at 55°C. Higher functional temperature of the mutant was attributable to the increase in thermostability of the gene product because recombinant protein produced from tsr(H258Y) was more thermostable than that from tsr. In fact, the tsr(H258Y) gene served as a selectable marker for plasmid transformation of G. kaustophilus. This new marker could facilitate complex genetic modification of G. kaustophilus and potentially other Geobacillus spp.

Thermoadaptation-directed evolution of chloramphenicol acetyltransferase in an error-prone thermophile using improved procedures.

Enhancing the thermostability of thermolabile enzymes extends their practical utility. We previously demonstrated that an error-prone thermophile derived from Geobacillus kaustophilus HTA426 can generate mutant genes encoding enzyme variants that are more thermostable than the parent enzyme. Here, we used this approach, termed as thermoadaptation-directed enzyme evolution, to increase the thermostability of the chloramphenicol acetyltransferase (CAT) of Staphylococcus aureus and successfully generated a CAT variant with an A138T replacement (CAT(A138T)). This variant was heterologously produced, and its enzymatic properties were compared with those of the wild type. We found that CAT(A138T) had substantially higher thermostability than CAT but had comparable activities, showing that the A138T replacement enhanced protein thermostability without affecting the catalytic activity. Because variants CAT(A138S) and CAT(A138V), which were generated via in vitro site-directed mutagenesis, were more thermostable than CAT, the thermostability enhancement resulting from the A138T replacement can be attributed to both the presence of a hydroxyl group and the bulk of the threonine side chain. CAT(A138T) conferred chloramphenicol resistance to G. kaustophilus cells at high temperature more efficiently than CAT. Therefore, the gene encoding CAT(A138T) may be useful as a genetic marker in Geobacillus spp. Notably, CAT(A138T) generation was achieved only by implementing improved procedures (plasmid-based mutations on solid media); previous procedures (chromosome-based mutations in liquid media) were unsuccessful. This result suggests that this improved procedure is crucial for successful thermoadaptation-directed evolution in certain cases and increases the opportunities for generating thermostable enzymes.

Active site loop conformation regulates promiscuous activity in a lactonase from Geobacillus kaustophilus HTA426.

Enzyme promiscuity is a prerequisite for fast divergent evolution of biocatalysts. A phosphotriesterase-like lactonase (PLL) from Geobacillus kaustophilus HTA426 (GkaP) exhibits main lactonase and promiscuous phosphotriesterase activities. To understand its catalytic and evolutionary mechanisms, we investigated a “hot spot” in the active site by saturation mutagenesis as well as X-ray crystallographic analyses. We found that position 99 in the active site was involved in substrate discrimination. One mutant, Y99L, exhibited 11-fold improvement over wild-type in reactivity (kcat/Km) toward the phosphotriesterase substrate ethyl-paraoxon, but showed 15-fold decrease toward the lactonase substrate δ-decanolactone, resulting in a 157-fold inversion of the substrate specificity. Structural analysis of Y99L revealed that the mutation causes a ∼6.6 Å outward shift of adjacent loop 7, which may cause increased flexibility of the active site and facilitate accommodation and/or catalysis of organophosphate substrate. This study provides for the PLL family an example of how the evolutionary route from promiscuity to specificity can derive from very few mutations, which promotes alteration in the conformational adjustment of the active site loops, in turn draws the capacity of substrate binding and activity.

Thermoadaptation-directed enzyme evolution in an error-prone thermophile derived from Geobacillus kaustophilus HTA426.

Thermostability is an important property of enzymes utilized for practical applications because it allows long-term storage and use as catalysts. In this study, we constructed an error-prone strain of the thermophile Geobacillus kaustophilus HTA426 and investigated thermoadaptation-directed enzyme evolution using the strain. A mutation frequency assay using the antibiotics rifampin and streptomycin revealed that G. kaustophilus had substantially higher mutability than Escherichia coli and Bacillus subtilis. The predominant mutations in G. kaustophilus were A · T→G · C and C · G→T · A transitions, implying that the high mutability of G. kaustophilus was attributable in part to high-temperature-associated DNA damage during growth. Among the genes that may be involved in DNA repair in G. kaustophilus, deletions of the mutSL, mutY, ung, and mfd genes markedly enhanced mutability. These genes were subsequently deleted to construct an error-prone thermophile that showed much higher (700- to 9,000-fold) mutability than the parent strain. The error-prone strain was auxotrophic for uracil owing to the fact that the strain was deficient in the intrinsic pyrF gene. Although the strain harboring Bacillus subtilis pyrF was also essentially auxotrophic, cells became prototrophic after 2 days of culture under uracil starvation, generating B. subtilis PyrF variants with an enhanced half-denaturation temperature of >10°C. These data suggest that this error-prone strain is a promising host for thermoadaptation-directed evolution to generate thermostable variants from thermolabile enzymes.

A novel thermostable β-galactosidase from Geobacillus kaustophilus HTA42

A novel thermostable β-galactosidase gene, designated as GkGal1A, from the thermophilic bacterium Geobacillus kaustophilus HTA426 was cloned and heterologously overexpressed in Escherichia coli(E. coli). Based on the sequence analysis, GkGal1A belongs to the glycosyl hydrolase family 1 that was the first β-galactosidase of bacterial origins expressed by us in this family. The apparent molecular weight of GkGal1A determined by sodium deodecyl sulfate-polyacrylamide gel electrophoresis is 52000. It exhibited the highest activity toward p-nitrophenyl-β-D-galactopyranoside at pH 7.8 and 70 °C and displayed high thermal stability. Divalent cations are prerequisite for the activity of GKGal1A, with the highest activity in the presence of Mn2+. Moreover, the three-dimensional structure of GkGal1A was modeled to speculate the structure of the catalytic residues and the reaction mechanism. The catalytic residues consisting of Glu166 and Glu355 were verified by site-directed mutagenesis.

Structural and functional characterization of the Geobacillus copper nitrite reductase: Involvement of the unique N-terminal region in the interprotein electron transfer with its redox partner

The crystal structures of copper-containing nitrite reductase (CuNiR) from the thermophilic Gram-positive bacterium Geobacillus kaustophilus HTA426 and the amino (N)-terminal 68 residue-deleted mutant were determined at resolutions of 1.3Å and 1.8Å, respectively. Both structures show a striking resemblance with the overall structure of the well-known CuNiRs composed of two Greek key β-barrel domains; however, a remarkable structural difference was found in the N-terminal region. The unique region has one β-strand and one α-helix extended to the northern surface of the type-1 copper site. The superposition of the Geobacillus CuNiR model on the electron-transfer complex structure of CuNiR with the redox partner cytochrome c551 in other denitrifier system led us to infer that this region contributes to the transient binding with the partner protein during the interprotein electron transfer reaction in the Geobacillus system. Furthermore, electron-transfer kinetics experiments using N-terminal residue-deleted mutant and the redox partner, Geobacillus cytochrome c551, were carried out. These structural and kinetics studies demonstrate that the region is directly involved in the specific partner recognition.

A Ternary Conjugation System for the Construction of DNA Libraries forGeobacillus kaustophilusHTA426

This report describes efficient plasmid uptake by the thermophile Geobacillus kaustophilus HTA426 by means of a ternary conjugation system, which was used to construct thermophile DNA libraries for G. kaustophilus and to identify the genes for orotidine-5'-phosphate decarboxylase by in vivo functional screening. The results indicate that the conjugation system is useful in constructing G. kaustophilus libraries, which are practical in identifying thermophile genes.

Ethanol production by In situ xylose isomerization using recombinant Escherichia coli and fermentation using conventional Saccharomyces cerevisiae

Xylose isomerase from Geobacillus kaustophilus HTA426 was functionally expressed in Escherichia coli BL21 (DE3) and the recombinant E. coli cells were used together with conventional Saccharomyces cerevisiae to produce ethanol from xylose by simultaneous xylose isomerisation and fermentation. When recombinant E. coli cells were used as the source of xylose isomerase, a significant amount of ethanol was produced from xylose, whereas the control without recombinant E. coli cells did not produce any detectable amount of ethanol from xylose. Ethanol production was increased by 38% by feeding more recombinant E. coli at 48 h compared to adding recombinant E. coli only in the beginning, resulting in more ethanol production than P. stipitis CBS6054 under the same conditions. The xylitol accumulation by the in situ process was only 57% of that produced by the P. stipitis CBS6054.

Genetic Transformation of Geobacillus kaustophilus HTA426 by Conjugative Transfer of Host-Mimicking Plasmids

We established an efficient transformation method for thermophile Geobacillus kaustophilus HTA426 using conjugative transfer from Escherichia coli of host-mimicking plasmids that imitate DNA methylation of strain HTA426 to circumvent its DNA restriction barriers. Two conjugative plasmids, pSTE33T and pUCG18T, capable of shuttling between E. coli and Geobacillus spp., were constructed. The plasmids were first introduced into E. coli BR408, which expressed one inherent DNA methylase gene (dam) and two heterologous methylase genes from strain HTA426 (GK1380-GK1381 and GK0343-GK0344). The plasmids were then directly transferred from E. coli cells to strain HTA426 by conjugative transfer using pUB307 or pRK2013 as a helper plasmid. pUCG18T was introduced very efficiently (transfer efficiency, 10(-5)-10(-3) recipient(-1)). pSTE33T showed lower efficiency (10(-7)-10(-6) recipient(-1)) but had a high copy number and high segregational stability. Methylase genes in the donor substantially affected the transfer efficiency, demonstrating that the host-mimicking strategy contributes to efficient transformation. The transformation method, along with the two distinguishing plasmids, increases the potential of G. kaustophilus HTA426 as a thermophilic host to be used in various applications and as a model for biological studies of this genus. Our results also demonstrate that conjugative transfer is a promising approach for introducing exogenous DNA into thermophiles.

Counterselection System for Geobacillus kaustophilus HTA426 through Disruption of pyrF and pyrR

Counterselection systems facilitate marker-free genetic modifications in microbes by enabling positive selections for both the introduction of a marker gene into the microbe and elimination of the marker from the microbe. Here we report a counterselection system for Geobacillus kaustophilus HTA426, established through simultaneous disruption of the pyrF and pyrR genes. The pyrF gene, essential for pyrimidine biosynthesis and metabolization of 5-fluoroorotic acid (5-FOA) to toxic metabolites, was disrupted by homologous recombination. The resultant MK54 strain (ΔpyrF) was auxotrophic for uracil and resistant to 5-FOA. MK54 complemented with pyrF was prototrophic for uracil but insensitive to 5-FOA in the presence of uracil. To confer 5-FOA sensitivity, the pyrR gene encoding an attenuator to repress pyrimidine biosynthesis by sensing uracil derivatives was disrupted. The resultant MK72 strain (ΔpyrF ΔpyrR) was auxotrophic for uracil and resistant to 5-FOA. MK72 complemented with pyrF was prototrophic for uracil and 5-FOA sensitive even in the presence of uracil. The results suggested that pyrF could serve as a counterselection marker in MK72, which was demonstrated by efficient marker-free integrations of heterologous β-galactosidase and α-amylase genes. The integrated genes were functionally expressed in G. kaustophilus and conferred new functions on the thermophile. This report describes the first establishment of a pyrF-based counterselection system in a Bacillus-related bacterium, along with the first demonstration of homologous recombination and heterologous gene expression in G. kaustophilus. Our results also suggest a new strategy for establishment of counterselection systems.

Crystal Structure of DnaK Protein Complexed with Nucleotide Exchange Factor GrpE in DnaK Chaperone System: INSIGHT INTO INTERMOLECULAR COMMUNICATION

The conserved, ATP-dependent bacterial DnaK chaperones process client substrates with the aid of the co-chaperones DnaJ and GrpE. However, in the absence of structural information, how these proteins communicate with each other cannot be fully delineated. For the study reported here, we solved the crystal structure of a full-length Geobacillus kaustophilus HTA426 GrpE homodimer in complex with a nearly full-length G. kaustophilus HTA426 DnaK that contains the interdomain linker (acting as a pseudo-substrate), and the N-terminal nucleotide-binding and C-terminal substrate-binding domains at 4.1-Å resolution. Each complex contains two DnaKs and two GrpEs, which is a stoichiometry that has not been found before. The long N-terminal GrpE α-helices stabilize the linker of DnaK in the complex. Furthermore, interactions between the DnaK substrate-binding domain and the N-terminal disordered region of GrpE may accelerate substrate release from DnaK. These findings provide molecular mechanisms for substrate binding, processing, and release during the Hsp70 chaperone cycle.