Production of 4-Deoxy-L-erythro-5-Hexoseulose Uronic Acid Using Two Free and Immobilized Alginate Lyases from Falsirhodobacter sp. Alg1.

Falsirhodobacter sp. alg1 expresses two alginate lyases, AlyFRA and AlyFRB, to produce the linear monosaccharide 4-deoxy-L-erythro-5-hexoseulose uronic acid (DEH) from alginate, metabolizing it to pyruvate. In this study, we prepared recombinant AlyFRA and AlyFRB and their immobilized enzymes and investigated DEH production. Purified AlyFRA and AlyFRB reacted with sodium alginate and yielded approximately 96.8% DEH. Immobilized AlyFRA and AlyFRB were prepared using each crude enzyme solution and κ-carrageenan, and immobilized enzyme reuse in batch reactions and DEH yield were examined. Thus, DEH was produced in a relatively high yield of 79.6%, even after the immobilized enzyme was reused seven times. This method can produce DEH efficiently and at a low cost and can be used to mass produce the next generation of biofuels using brown algae.

Cell-Penetrating Peptide-Peptide Nucleic Acid Conjugates as a Tool for Protein Functional Elucidation in the Native Bacterium.

Approximately 30% or more of the total proteins annotated from sequenced bacteria genomes are annotated as hypothetical or uncharacterized proteins. However, elucidation on the function of these proteins is hindered by the lack of simple and rapid screening methods, particularly with novel or hard-to-transform bacteria. In this report, we employed cell-penetrating peptide (CPP) -peptide nucleotide acid (PNA) conjugates to elucidate the function of such uncharacterized proteins in vivo within the native bacterium. Paenibacillus, a hard-to-transform bacterial genus, was used as a model. Two hypothetical genes showing amino acid sequence similarity to ι-carrageenases, termed cgiA and cgiB, were identified from the draft genome of Paenibacillus sp. strain YYML68, and CPP-PNA probes targeting the mRNA of the acyl carrier protein gene, acpP, and the two ι-carrageenase candidate genes were synthesized. Upon direct incubation of CPP-PNA targeting the mRNA of the acpP gene, we successfully observed growth inhibition of strain YYML68 in a concentration-dependent manner. Similarly, both the function of the candidate ι-carrageenases were also inhibited using our CPP-PNA probes allowing for the confirmation and characterization of these hypothetical proteins. In summary, we believe that CPP-PNA conjugates can serve as a simple and efficient alternative approach to characterize proteins in the native bacterium.

Single-bacterial genomics validates rich and varied specialized metabolism of uncultivated Entotheonella sponge symbionts.

Marine sponges are prolific sources of unique bioactive natural products. The sponge Theonella swinhoei is represented by several distinct variants with largely nonoverlapping chemistry. For the Japanese chemotype Y harboring diverse complex polyketides and peptides, we previously provided genomic and functional evidence that a single symbiont, the filamentous, multicellular organism "Candidatus Entotheonella factor," produces almost all of these compounds. To obtain further insights into the chemistry of "Entotheonella," we investigated another phylotype, "Candidatus Entotheonella serta," present in the T. swinhoei WA sponge chemotype, a source of theonellamide- and misakinolide-type compounds. Unexpectedly, considering the lower chemical diversity, sequencing of individual bacterial filaments revealed an even larger number of biosynthetic gene regions than for Ca E. factor, with virtually no overlap. These included genes for misakinolide and theonellamide biosynthesis, the latter assigned by comparative genomic and metabolic analysis of a T. swinhoei chemotype from Israel, and by biochemical studies. The data suggest that both compound families, which were among the earliest model substances to study bacterial producers in sponges, originate from the same bacterium in T. swinhoei WA. They also add evidence that metabolic richness and variability could be a more general feature of Entotheonella symbionts.

An environmental bacterial taxon with a large and distinct metabolic repertoire.

Cultivated bacteria such as actinomycetes are a highly useful source of biomedically important natural products. However, such 'talented' producers represent only a minute fraction of the entire, mostly uncultivated, prokaryotic diversity. The uncultured majority is generally perceived as a large, untapped resource of new drug candidates, but so far it is unknown whether taxa containing talented bacteria indeed exist. Here we report the single-cell- and metagenomics-based discovery of such producers. Two phylotypes of the candidate genus 'Entotheonella' with genomes of greater than 9 megabases and multiple, distinct biosynthetic gene clusters co-inhabit the chemically and microbially rich marine sponge Theonella swinhoei. Almost all bioactive polyketides and peptides known from this animal were attributed to a single phylotype. 'Entotheonella' spp. are widely distributed in sponges and belong to an environmental taxon proposed here as candidate phylum 'Tectomicrobia'. The pronounced bioactivities and chemical uniqueness of 'Entotheonella' compounds provide significant opportunities for ecological studies and drug discovery.

SAG-QC: quality control of single amplified genome information by subtracting non-target sequences based on sequence compositions.

BACKGROUND: Whole genome amplification techniques have enabled the analysis of unexplored genomic information by sequencing of single-amplified genomes (SAGs). Whole genome amplification of single bacteria is currently challenging because contamination often occurs in experimental processes. Thus, to increase the confidence in the analyses of sequenced SAGs, bioinformatics approaches that identify and exclude non-target sequences from SAGs are required. Since currently reported approaches utilize sequence information in public databases, they have limitations when new strains are the targets of interest. Here, we developed a software SAG-QC that identify and exclude non-target sequences independent of database. RESULTS: In our method, "no template control" sequences acquired during WGA were used. We calculated the probability that a sequence was derived from contaminants by comparing k-mer compositions with the no template control sequences. Based on the results of tests using simulated SAG datasets, the accuracy of our method for predicting non-target sequences was higher than that of currently reported techniques. Subsequently, we applied our tool to actual SAG datasets and evaluated the accuracy of the predictions. CONCLUSIONS: Our method works independently of public sequence information for distinguishing SAGs from non-target sequences. This method will be effective when employed against SAG sequences of unexplored strains and we anticipate that it will contribute to the correct interpretation of SAGs.

Metabolic and evolutionary origin of actin-binding polyketides from diverse organisms.

Actin-targeting macrolides comprise a large, structurally diverse group of cytotoxins isolated from remarkably dissimilar micro- and macroorganisms. In spite of their disparate origins and structures, many of these compounds bind actin at the same site and exhibit structural relationships reminiscent of modular, combinatorial drug libraries. Here we investigate biosynthesis and evolution of three compound groups: misakinolides, scytophycin-type compounds and luminaolides. For misakinolides from the sponge Theonella swinhoei WA, our data suggest production by an uncultivated 'Entotheonella' symbiont, further supporting the relevance of these bacteria as sources of bioactive polyketides and peptides in sponges. Insights into misakinolide biosynthesis permitted targeted genome mining for other members, providing a cyanobacterial luminaolide producer as the first cultivated source for this dimeric compound family. The data indicate that this polyketide family is bacteria-derived and that the unusual macrolide diversity is the result of combinatorial pathway modularity for some compounds and of convergent evolution for others.

Construction of bioengineered yeast platform for direct bioethanol production from alginate and mannitol.

Brown macroalgae are a sustainable and promising source for bioethanol production because they are abundant in ocean ecosystems and contain negligible quantities of lignin. Brown macroalgae contain cellulose, hemicellulose, mannitol, laminarin, and alginate as major carbohydrates. Among these carbohydrates, brown macroalgae are characterized by high levels of alginate and mannitol. The direct bioconversion of alginate and mannitol into ethanol requires extensive bioengineering of assimilation processes in the standard industrial microbe Saccharomyces cerevisiae. Here, we constructed an alginate-assimilating S. cerevisiae recombinant strain by genome integration and overexpression of the genes encoding endo- and exo-type alginate lyases, DEH (4-deoxy-L-erythro-5-hexoseulose uronic acid) transporter, and components of the DEH metabolic pathway. Furthermore, the mannitol-metabolizing capacity of S. cerevisiae was enhanced by prolonged culture in a medium containing mannitol as the sole carbon source. When the constructed strain AM1 was anaerobically cultivated in a fermentation medium containing 6% (w/v) total sugars (approximately 1:2 ratio of alginate/mannitol), it directly produced ethanol from alginate and mannitol, giving 8.8 g/L ethanol and yields of up to 32% of the maximum theoretical yield from consumed sugars. These results indicate that all major carbohydrates of brown macroalgae can be directly converted into bioethanol by S. cerevisiae. This strain and system could provide a platform for the complete utilization of brown macroalgae.

Formosa haliotis sp. nov., a brown-alga-degrading bacterium isolated from the gut of the abalone Haliotis gigantea.

Four brown-alga-degrading, Gram-stain-negative, aerobic, non-flagellated, gliding and rod-shaped bacteria, designated LMG 28520T, LMG 28521, LMG 28522 and LMG 28523, were isolated from the gut of the abalone Haliotis gigantea obtained in Japan. The four isolates had identical random amplified polymorphic DNA patterns and grew optimally at 25 °C, at pH 6.0-9.0 and in the presence of 1.0-4.0 % (w/v) NaCl. Phylogenetic trees based on 16S rRNA gene sequences placed the isolates in the genus Formosa with Formosa algae and Formosa arctica as closest neighbours. LMG 28520T and LMG 28522 showed 100 % DNA-DNA relatedness to each other, 16-17 % towards F. algae LMG 28216T and 17-20 % towards F. arctica LMG 28318T; they could be differentiated phenotypically from these established species. The predominant fatty acids of isolates LMG 28520T and LMG 28522 were summed feature 3 (iso-C15 : 0 2-OH and/or C16 : 1ω7c), iso-C15 : 1 G and iso-C15 : 0. Isolate LMG 28520T contained menaquinone-6 (MK-6) as the major respiratory quinone and phosphatidylethanolamine, two unknown aminolipids and an unknown lipid as the major polar lipids. The DNA G+C content was 34.4 mol% for LMG 28520T and 35.5 mol% for LMG 28522. On the basis of their phylogenetic and genetic distinctiveness, and differential phenotypic properties, the four isolates are considered to represent a novel species of the genus Formosa, for which the name Formosa haliotis sp. nov. is proposed. The type strain is LMG 28520T ( = NBRC 111189T).

In situ detection of antibiotic amphotericin B produced in Streptomyces nodosus using Raman microspectroscopy.

The study of spatial distribution of secondary metabolites within microbial cells facilitates the screening of candidate strains from marine environments for functional metabolites and allows for the subsequent assessment of the production of metabolites, such as antibiotics. This paper demonstrates the first application of Raman microspectroscopy for in situ detection of the antifungal antibiotic amphotericin B (AmB) produced by actinomycetes-Streptomyces nodosus. Raman spectra measured from hyphae of S. nodosus show the specific Raman bands, caused by resonance enhancement, corresponding to the polyene chain of AmB. In addition, Raman microspectroscopy enabled us to monitor the time-dependent change of AmB production corresponding to the growth of mycelia. The Raman images of S. nodosus reveal the heterogeneous distribution of AmB within the mycelia and individual hyphae. Moreover, the molecular association state of AmB in the mycelia was directly identified by observed Raman spectral shifts. These findings suggest that Raman microspectroscopy could be used for in situ monitoring of antibiotic production directly in marine microorganisms with a method that is non-destructive and does not require labeling.

Draft Genome of Kangiella sp. Strain TOML190, Isolated from the Surface of the Striped Shore Crab, Pachygrapsus crassipes.

Kangiella sp. strain TOML190 is a strain from the Kangiella genus that was isolated from the surface of a crustacean. Genetic background analysis of this strain shows that it harbors unique features possibly related to its symbiotic adaptation to its residing host.

Changes in Tissue Distribution of Tetrodotoxin and Its Analogues in Association with Maturation in the Toxic Flatworm, Planocera multitentaculata.

The toxic flatworm, Planocera multitentaculata, possesses highly concentrated tetrodotoxin (TTX), also known as pufferfish toxin, throughout its life cycle, including the egg and larval stages. Additionally, TTX analogues, 5,6,11-trideoxyTTX and 11-norTTX-6(S)-ol, have also been detected in the flatworm. The high concentration of TTX in the eggs and larvae appears to be for protection against predation, and 11-norTTX-6(S)-ol in the pharyngeal tissue in the adults is likely used to sedate or kill prey during predation. However, information on the role of 5,6,11-trideoxyTTX, a potential important biosynthetic intermediate of TTX, in the toxic flatworm is lacking. Here, we aimed to determine the region of localization of TTX and its analogues in the flatworm body, understand their pharmacokinetics during maturation, and speculate on their function. Flatworm specimens in four stages of maturity, namely juvenile, mating, spawning, and late spawning, were subjected to LC-MS/MS analysis, using the pharyngeal tissue, oocytes in seminal receptacle, sperm, and tissue from 12 other sites. Although TTX was consistently high in the pharyngeal tissue throughout maturation, it was extremely high in the oocytes during the spawning period. Meanwhile, 5,6,11-trideoxyTTX was almost undetectable in the pharyngeal part throughout the maturation but was very abundant in the oocytes during spawning. 11-norTTX-6(S)-ol consistently localized in the pharyngeal tissue. Although the localization of TTX and its analogues was approximately consistent with the MS imaging data, TTX and 11-norTTX-6(S)-ol were found to be highly localized in the parenchyma surrounding the pharynx, which suggests the parenchyma is involved in the accumulation and production of TTXs.

Single-cell metabolite detection and genomics reveals uncultivated talented producer.

The production of bioactive metabolites is increasingly recognized as an important function of host-associated bacteria. An example is defensive symbiosis that might account for much of the chemical richness of marine invertebrates including sponges (Porifera), 1 of the oldest metazoans. However, most bacterial members of sponge microbiomes have not been cultivated or sequenced, and therefore, remain unrecognized. Unequivocally linking metabolic functions to a cellular source in sponge microbiomes is, therefore, a challenge. Here, we report an analysis pipeline of microfluidic encapsulation, Raman microscopy, and integrated digital genomics (MERMAID) for an efficient identification of uncultivated producers. We applied this method to the chemically rich bacteriosponge (sponge that hosts a rich bacterial community) Theonella swinhoei, previously shown to contain 'Entotheonella' symbionts that produce most of the bioactive substances isolated from the sponge. As an exception, the antifungal aurantosides had remained unassigned to a source. Raman-guided single-bacterial analysis and sequencing revealed a cryptic, distinct multiproducer, 'Candidatus Poriflexus aureus' from a new Chloroflexi lineage as the aurantoside producer. Its exceptionally large genome contains numerous biosynthetic loci and suggested an even higher chemical richness of this sponge than previously appreciated. This study highlights the importance of complementary technologies to uncover microbiome functions, reveals remarkable parallels between distantly related symbionts of the same host, and adds functional support for diverse chemically prolific lineages being present in microbial dark matter.

Microfluidic device with chemical gradient for single-cell cytotoxicity assays.

Here, we report the fabrication of a chemical gradient microfluidic device for single-cell cytotoxicity assays. This device consists of a microfluidic chemical gradient generator and a microcavity array that enables entrapment of cells with high efficiency at 88 ± 6% of the loaded cells. A 2-fold logarithmic chemical gradient generator that is capable of generating a serial 2-fold gradient was designed and then integrated with the microcavity array. High density single-cell entrapment was demonstrated in the device without cell damage, which was performed in 30 s. Finally, we validated the feasibility of this device to perform cytotoxicity assays by exposing cells to potassium cyanide (0-100 µM KCN). The device captured images of 4000 single cells affected by 6 concentrations of KCN and determined cell viability by counting the effected cells. Image scanning of the microcavity array was completed within 10 min using a 10× objective lens and a motorized stage. Aligning cells on the microcavity array eases cell counting, observation, imaging, and evaluation of singular cells. Thus, this platform was able to determine the cytotoxicity of chemicals at a single-cell level, as well as trace the cytotoxicity over time. This device and method will be useful for cytotoxicity analysis and basic biomedical research.

Critical Side Chain Effects of Cell-Penetrating Peptides for Transporting Oligo Peptide Nucleic Acids in Bacteria.

Of various methods for delivering functional molecules into cells, a chemical approach using cell-penetrating peptides (CPPs) is facile and highly efficient. Currently, however, there are few examples of CPPs highly efficient with bacteria in contrast to CPPs targeting animal cells, and thus our understanding of the structural effects of these bacteria-efficient CPPs, termed as BCPPs, on permeation efficiency is limited. Herein, we report a comprehensive investigation on the permeation efficiencies of cationic short peptides through bacterial cell membranes. We observed that elongating the length of the main chain increased permeation efficiency. More interestingly, the length of the peptide side chain critically affected permeation efficiency; shortening the side chain significantly enhanced efficiency. Among the BCPPs investigated, 2,3-diaminopropionic acid nonamer showed the highest permeation efficiency into bacterial cells of diverse strains, allowing the transport of oligo peptide nucleic acids and subsequent growth inhibition. This study provides insights into the molecular design of efficient BCPPs for manipulating bacterial growth.

Microbial community analysis in the gills of abalones suggested possible dominance of epsilonproteobacterium in Haliotis gigantea.

Gills are important organs for aquatic invertebrates because they harbor chemosynthetic bacteria, which fix inorganic carbon and/or nitrogen and provide their hosts with organic compounds. Nevertheless, in contrast to the intensive researches related to the gut microbiota, much is still needed to further understand the microbiota within the gills of invertebrates. Using abalones as a model, we investigated the community structure of microbes associated with the gills of these invertebrates using next-generation sequencing. Molecular identification of representative bacterial sequences was performed using cloning, nested PCR and fluorescence in situ hybridization (FISH) analysis with specific primers or probes. We examined three abalone species, namely Haliotis gigantea, H. discus and H. diversicolor using seawater and stones as controls. Microbiome analysis suggested that the gills of all three abalones had the unclassified Spirochaetaceae (one OTU, 15.7 ± 0.04%) and Mycoplasma sp. (one OTU, 9.1 ± 0.03%) as the core microbes. In most libraries from the gills of H. gigantea, however, a previously unknown epsilonproteobacterium species (one OTU) was considered as the dominant bacterium, which accounted for 62.2% of the relative abundance. The epsilonproteobacterium was only detected in the gills of H. diversicolor at 0.2% and not in H. discus suggesting that it may be unique to H. gigantea. Phylogenetic analysis performed using a near full-length 16S rRNA gene placed the uncultured epsilonproteobacterium species at the root of the family Helicobacteraceae. Interestingly, the uncultured epsilonproteobacterium was commonly detected from gill tissue rather than from the gut and foot tissues using a nested PCR assay with uncultured epsilonproteobacterium-specific primers. FISH analysis with the uncultured epsilonproteobacterium-specific probe revealed that probe-reactive cells in H. gigantea had a coccus-like morphology and formed microcolonies on gill tissue. This is the first report to show that epsilonproteobacterium has the potential to be a dominant species in the gills of the coastal gastropod, H. gigantea.

Taxonomic Distribution of Tetrodotoxin in Acotylean Flatworms (Polycladida: Platyhelminthes).

Tetrodotoxin (TTX), also known as pufferfish toxin, causes a respiratory disorder by blocking neurotransmission, with voltage-gated sodium channel inhibition on muscle and nerve tissues. The toxin is widely distributed across vertebrates, invertebrates and bacteria. Therefore, it is generally thought that TTX in pufferfish accumulates via the food webs, beginning with marine bacteria as a primary producer. Polyclad flatworms in the genus Planocera are also known to be highly toxic, TTX-bearing organisms. Unlike the case of pufferfish, the source of TTX in these flatworms is unknown. In this study, taxonomical distribution patterns of TTX were investigated for acotylean flatworms from coastal waters using molecular phylogenetic analysis and high performance liquid chromatography-tandem mass spectrometry (LC-MS/MS). A maximum likelihood tree based on the 28S rRNA gene sequence showed that the flatworms belonged to several different lineages among the genera Planocera, Stylochus, Paraplanocera, Discocelis, Notocomplana, Notoplana, Callioplana and Peudostylochus. After LC-MS/MS analysis, the distribution of TTX was mapped onto the molecular phylogenetic tree. TTX-bearing flatworm species were seen to be restricted to specific Planocera lineages, suggesting that the TTX-bearing flatworm species have common genes for TTX-accumulating mechanisms.

High-density microcavity array for cell detection: single-cell analysis of hematopoietic stem cells in peripheral blood mononuclear cells.

Detection and isolation of specific cell types from limited biological samples have become a major challenge in clinical diagnosis and cell biology research. Here, we report a high-density microcavity array for target cell detection in which thousands of single cells were neatly arrayed onto 10,000 microcavities with high efficiency at approximately 90% of the loaded cells. Cell-specific immunophenotypes were exclusively identified at the single-cell level by measuring fluorescence intensities of cells labeled with antibodies targeting cell surface markers, and the purity of hematopoietic stem cells (HSCs) within human peripheral blood analyzed by this system was correlated with those obtained by conventional flow cytometry. Furthermore, gene expression of the stem cell marker, CD34, was determined from HSCs by isolating single cells using a micromanipulator. This technology has proven to be an effective tool for target cell detection and subsequent cellular analytical research at the single-cell level.

Nano-sized bacterial magnetic particles displaying pyruvate phosphate dikinase for pyrosequencing.

There is a high demand for inexpensive and high-throughput DNA sequencing technologies in molecular biology and applied biosciences. In this study, novel nano-sized magnetic particles displaying enzymes for pyrosequencing, a rather novel bioluminometric DNA sequencing method based on the sequencing-by-synthesis principle by employing a cascade of several enzymatic reactions, was developed. A highly thermostable enzyme, pyruvate phosphate dikinase (PPDK) which converts PPi to ATP was successfully expressed onto bacterial magnetic particles (BacMPs) using a novel protein display system of Magnetospirillum magneticum AMB-1. The enzymatic stability of BacMPs displaying PPDK (PPDK-BacMPs) to pH and temperature was evaluated and its broad range of properties was shown. Subsequently, PPDK-BacMPs were applied in pyrosequencing and a target oligonucleotide was successfully sequenced. The PPDK enzyme displayed on BacMPs was shown to be recyclable in each sequence reaction as they can be manipulated by magnetic force. It was concluded that nano-sized PPDK-BacMPs are useful for the scale down of pyrosequencing reaction volumes, thus, permitting high-throughput. The recycling of enzymes was also shown to be promising and applicable for the development of an inexpensive DNA sequencing at a low running cost.

A stable human progesterone receptor expressing HeLa reporter cell line as a tool in chemical evaluation at the different cell-cycle phases.

Specific molecular events, characteristic of each cell-cycle phase may have direct effect to the functionality of nuclear receptors. Based on this understanding, the evaluation of lipophilic chemicals at the different cell-cycle phases is significant and should be considered. In order to achieve the aim of performing large-scale dose-response analysis on the effects of lipophilic chemicals at the different cell-cycle phases, a stable, sensitive and highly selective human progesterone receptor (hPR) expressing HeLa reporter cell line, hPRLuc-20, was established. Upon the establishment of the hPRLuc-20 cells, they were synchronized to the G(1), S and G(2) phases and treated with progesterone (PROG) and promegestone (R5020). The cells successfully showed that at the different cell-cycle phase, both agonists resulted in different cellular responses. The differences in response supports that hPR expressed within the hPRLuc-20 cells do respond in a cell-cycle dependent manner, thus showing the cells' compatibility in large-scale dose-response analyses of chemicals. It is hopeful that the advanced application of the hPRLuc-20 cells could contribute to provide fundamental hints to further understand the function of hPR, and provide key observations to elucidate the nature of these chemicals with hPR, its corresponding co-regulators and transcription factors.

Abiotic Factors Promote Cell Penetrating Peptide Permeability in Enterobacteriaceae Models.

Conventionally, the delivery of biomolecules into bacteria for the generation of characterized or functional mutants has relied greatly on horizontal gene transfer techniques. However, the low compatibility of these techniques with novel or hard-to-transform bacteria currently serves as a challenge to the bioengineering field. Here, we explored the use of cell penetrating peptides (CPPs) as an alternative biomolecule delivery approach by investigating the effects of the abiotic factors during CPP permeation. Using the (KFF)3K-FAM conjugate and Escherichia coli as models, we evaluated four abiotic factors where two of these factors, temperature and solution tonicity, promoted (KFF)3K-FAM permeation efficiency. Our data show that optimal (KFF)3K-FAM permeation efficiency was achieved for E. coli at approximately 98.1% under conditions of 37°C (growth optimal temperature) and 50% PBS concentration. Based on these conditions, we subsequently tested the applicability of CPP permeation in various bacterial strains by treating 10 bacterial strains from the Enterobacteriaceae family among which seven strains have no CPP permeation records with (KFF)3K-FAM. Interestingly, when compared with non-optimized conditions, all 10 strains showed a marked increase in CPP permeation ranging between 20 and 90% efficiency. Although using strains within Enterobacteriaceae that are phylogenetically close, our results hinted on the possibility that with proper optimization of the abiotic factors, CPPs could be compatible with a broad range of bacterial strains. Our efforts suggest that CPP could serve as an effective alternative approach for mutant generation and for biomolecule delivery into novel or hard-to-transform bacteria.