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Background: Plasmid-borne genetic editing tools, including the widely used CRISPR-Cas9 system, have greatly facilitated bacterial programming to obtain novel functionalities. However, the lack of effective post-editing plasmid elimination methods impedes follow-up genetic manipulation or application. Conventional strategies including exposure to physical and chemical treatments, or exploiting temperature-sensitive replication origins have several drawbacks (e.g., they are limited for efficiency and are time-consuming). Therefore, the demand is apparent for easy and rapid elimination of the tool plasmids from their bacterial hosts after genetic manipulation. Results: To bridge this gap, we designed a novel EXIT circuit with the homing endonuclease, which can be exploited for rapid and efficient elimination of various plasmids with diverse replication origins. As a proof of concept, we validated the EXIT circuit in Escherichia coli by harnessing homing endonuclease I-SceI and its cleavage site. When integrated into multiple plasmids with different origins, the EXIT circuit allowed them to be eliminated from the host cells, simultaneously. By combining the widely used plasmid-borne CRISPR-Cas9 system and the EXIT circuit, we constructed an easy-to-use CRISPR-Cas9 system that eliminated the Cas9- and the single-guide RNA (sgRNA)-encoding plasmids in one-step. Within 3 days, we successfully constructed an atrazine-degrading E. coli strain, thus further demonstrating the advantage of this new CRISPR-Cas9 system for bacterial genome editing. Conclusions: Our novel EXIT circuit, which exploits the homing endonuclease I-SceI, enables plasmid(s) with different replication origins to be eliminated from their host cells rapidly and efficiently. We also developed an easy-to-use CRISPR-Cas9 system with the EXIT circuit, and this new system can be widely applied to bacterial genome editing. [ABSTRACT FROM AUTHOR]

This paper describes the microfluidic streak plate (MSP), a facile method for high-throughput microbial cell separation and cultivation in nanoliter sessile droplets. The MSP method builds upon the conventional streak plate technique by using microfluidic devices to generate nanoliter droplets that can be streaked manually or robotically onto petri dishes prefilled with carrier oil for cultivation of single cells. In addition, chemical gradients could be encoded in the droplet array for comprehensive dose-response analysis. The MSP method was validated by using single-cell isolation of Escherichia coli and antimicrobial susceptibility testing of Pseudomonas aeruginosa PAO1. The robustness of the MSP work flow was demonstrated by cultivating a soil community that degrades polycyclic aromatic hydrocarbons. Cultivation in droplets enabled detection of the richest species diversity with better coverage of rare species. Moreover, isolation and cultivation of bacterial strains by MSP led to the discovery of several species with high degradation efficiency, including four Mycobacterium isolates and a previously unknown fluoranthene-degrading Blastococcus species. [ABSTRACT FROM AUTHOR]

Conserved clusters of genes encoding DsrE and TusA homologs occur in many archaeal and bacterial sulfur oxidizers. TusA has a well documented function as a sulfurtransferase in tRNA modification and molybdenum cofactor biosynthesis in Escherichia coli, and DsrE is an active site subunit of the DsrEFH complex that is essential for sulfur trafficking in the pho-totrophic sulfur-oxidizing Allochromatium vinosum. In the acidothermophilic sulfur (S0)- and tetrathionate (S4O2-6)-oxidizing Metallosphaera cuprina Ar-4, a dsrE3A-dsrE2B-tusA arrangement is situated immediately between genes encoding dihydrolipoamide dehydrogenase and a heterodisulfide reductase-like complex. In this study, the biochemical features and sulfur transferring abilities of the DsrE2B, DsrE3A, and TusA proteins were investigated. DsrE3A and TusA proved to react with tetrathionate but not with NaSH, glutathione persulfide, polysulfide, thiosulfate, or sulfite. The products were identified as protein-Cys-S-thiosulfonates. DsrE3A was also able to cleave the thiosulfate group from TusA-Cys18-S-thiosulfonate. DsrE2B did not react with any of the sulfur compounds tested. DsrE3A and TusA interacted physically with each other and formed a heterocomplex. The cysteine residue (Cys18) of TusA is crucial for this interaction. The single cysteine mutants DsrE3A-C93 and DsrE3A-C101S retained the ability to transfer the thiosulfonate group to TusA. TusA-C18S neither reacted with tetrathionate nor was it loaded with thiosulfate with DsrE3A-Cys-S-thiosulfonate as the donor. The transfer of thiosulfate, mediated by a DsrE-like protein and TusA, is unprecedented not only in M. cuprina but also in other sulfur-oxidizing prokaryotes. The results of this study provide new knowledge on oxidative microbial sulfur metabolism. [ABSTRACT FROM AUTHOR]

Corynebacterium glutamicum is widely used for industrial production of various amino acids and vitamins, and there is growing interest in engineering this bacterium for more commercial bioproducts such as -aminobutyric acid (GABA). In this study, a C. glutamicum GABA-specific transporter (GabP Cg ) encoded by ncgl0464 was identified and characterized. GabP Cg plays a major role in GABA uptake and is essential to C. glutamicum growing on GABA. GABA uptake by GabP Cg was weakly competed by l-Asn and l-Gln and stimulated by sodium ion (Na+). The Km and V max values were determined to be 41.1 ± 4.5 μM and 36.8 ± 2.6 nmol min–1 (mg dry weight [DW])-1, respectively, at pH 6.5 and 34.2 ± 1.1 μM and 67.3 ± 1.0 nmol min-1 (mg DW)-1, respectively, at pH 7.5. GabP Cg has 29% amino acid sequence identity to a previously and functionally identified aromatic amino acid transporter (TyrP) of Escherichia coli but low identities to the currently known GABA transporters (17% and 15% to E. coli GabP and Bacillus subtilis GabP, respectively). The mutant RES167 ncg10464/pGXKZ9 with the GabP Cg deletion showed 12.5% higher productivity of GABA than RES167/pGXKZ9. It is concluded that GabP Cg represents a new type of GABA transporter and is potentially important for engineering GABA-producing C. glutamicum strains. [ABSTRACT FROM AUTHOR]

Corynebacterium glutamicum grew on resorcinol as a sole source of carbon and energy. By genome-wide data mining, two gene clusters, designated NCgl1110–NCgl1113 and NCg12950–NCg12953, were proposed to encode putative proteins involved in resorcinol catabolism. Deletion of the NCg12950–NCg12953 gene cluster did not result in any observable phenotype changes. Disruption and complementation of each gene at NCgl1110–NCgl1113, NCg12951, and NCg12952 indicated that these genes were involved in resorcinol degradation. Expression of NCgl1112, NCgl1113, and NCg12951 in Escherichia coli revealed that NCgl1113 and NCg12951 both coded for hydroxyquinol 1,2-dioxygenases and NCgl1112 coded for maleylacetate reductases. NCgl1111 encoded a putative monooxygenase, but this putative hydroxylase was very different from previously functionally identified hydroxylases. Cloning and expression of NCgl1111 in E. coli revealed that NCgl1111 encoded a resorcinol hydroxylase that needs NADPH as a cofactor. E. coli cells containing Ncgl1111 and Ncgl1113 sequentially converted resorcinol into maleylacetate. NCgl1110 and NCg12950 both encoded putative TetR family repressors, but only NCgl1110 was transcribed and functional. NCg12953 encoded a putative transporter, but disruption of this gene did not affect resorcinol degradation by C. glutamicum. The function of NCg12953 remains unclear. [ABSTRACT FROM AUTHOR]

Comamonas sp. strain CNB-1 grows on 4-chloronitrobenzene (4-CNB) and nitrobenzene as sole carbon and nitrogen sources. In this study, two genetic segments, cnbB-orf2-cnbA and cnbR-orf1-cnbCaCbDEFGHI, located on a newly isolated plasmid, pCNB1 (ca. 89 kb), and involved in 4-CNB/nitrobenzene degradation, were characterized. Seven genes (cnbA, cnbB, cnbCa, cnbCb, cnbD, cnbG, and cnbH) were cloned and functionally expressed in recombinant Escherichia coli, and they were identified as encoding 4-CNB nitroreductase (CnbA), 1-hydroxylaminobenzene mutase (CnbB), 2-aminophenol 1,6-dioxygenase (Cnb- Cab), 2-amino-5-chloromuconic semialdehyde dehydrogenase (CnbD), 2-hydroxy-5-chloromuconic acid (2H5CM) tautomerase, and 2-amino-5-chloromuconic acid (2A5CM) deaminase (CnbH). In particular, the 2A5CM deaminase showed significant identities (31 to 38%) to subunit A of Asp-tRNAAsn/Glu-tRNAGln amidotransferase and not to the previously identified deaminases for nitroaromatic compound degradation. Genetic cloning and expression of cnbH in Escherichia coli revealed that CnbH catalyzed the conversion of 2A5CM into 2H5CM and ammonium. Four other genes (cnbR, cnbE, cnbF, and cabI) were tentatively identified according to their high sequence identities to other functionally identified genes. It was proposed that CnbH might represent a novel type of deaminase and be involved in a novel partial reductive pathway for chloronitrobenzene or nitrobenzene degradation. [ABSTRACT FROM AUTHOR]

Corynebacterium glutamicum used gentisate and 3-hydroxybenzoate as its sole carbon and energy source for growth. By genome-wide data mining, a gene cluster designated ncg12918-ncg12923 was proposed to encode putative proteins involved in gentisate/3-hydroxybenzoate pathway. Genes encoding gentisate l,2-dioxygenase (ncg12920) and fumarylpyruvate hydrolase (ncg12919) were identified by cloning and expression of each gene in Escherichki coli. The gene of ncg12918 encoding a hypothetical protein (Ncg12918) was proved to be essential for gentisate-3-hydroxybenzoate assimilation. Mutant strain RES167Δncg12918 lost the ability to grow on gentisate or 3-hydroxybenzoate, but this ability could be restored in C. glutamicum upon the complementation with pXMJ19-ncg12919. Cloning and expression of this ncg12918 gene in E. coli showed that Ncg12918 is a glutathione-independent maleylpyruvate isomerase. Upstream of ncg12920, the genes ncg12921-ncg12923 were located, which were essential for gentisate and/or 3-hydroxybenzoate catabolism. The Ncg12921 was able to up-regulate gentisate 1,2-dioxygenase, maleylpyruvate isomerase, and fumarylpyruvate hydrolase activities. The genes ncg12922 and ncg12923 were deduced to encode a gentisate transporter protein and a 3-hydroxy- benzoate hydroxylase, respectively, and were essential for gentisate or 3-hydroxybenzoate assimilation. Based on the results obtained in this study, a GSII-independent gentis ate pathway was proposed, and genes involved in this pathway were identified. [ABSTRACT FROM AUTHOR]

The sulfur oxygenase/reductase (SOR) of Acidianus strain S5 was purified and characterized after expressing the SOR gene in a recombinant strain of Escherichia coli. The N-terminal sequence of the purified SOR protein was the same as the deduced amino acid sequence from previously cloned SOR genes. Enzymatic studies indicated that the SOR catalyzed the conversion of elemental sulfur (So) to sulfite, thiosulfate, and sulfide. The optimal pH and temperature were 5.0 and 70 °C, respectively. Comparison of this SOR and that of A. ambivalens revealed several differences between these two SORs. The most striking difference is that the SOR of Acidianus S5 had maximal activity at acidic pH. By application of anti-SOR serum and the Western blot technique, it was found that SOR proteins existed in A. brierleyi and in Acidianus S5 cells cultivated with thiosulfate as the sole energy source, indicating that SOR may also play a role in thiosulfate metabolism. [ABSTRACT FROM AUTHOR]