Reconsideration of the previously classified incompatibility groups of plasmids, IncP-1 and IncP-11.

This study presents the reassessment of earlier published data with reference to the article published in Environmental Microbiology entitled 'IncP-type plasmids carrying genes for antibiotic resistance or aromatic compound degradation are prevalent in sequenced Aromatoleum and Thauera strains' by Lo et al. This correspondence clarifies misperceptions of plasmids classified under incompatibility (Inc) groups IncP-1 and IncP-11.

Integrons, transposons and IS elements promote diversification of multidrug resistance plasmids and adaptation of their hosts to antibiotic pollutants from pharmaceutical companies.

Plasmids are important vehicles for the dissemination of antibiotic resistance genes (ARGs) among bacteria by conjugation. Here, we determined the complete nucleotide sequences of nine different plasmids previously obtained by exogenous plasmid isolation from river and creek sediments and wastewater from a pharmaceutical company. We identified six IncP/P-1ε plasmids and single members of IncL, IncN and IncFII-like plasmids. Genetic structures of the accessory regions of the IncP/P-1ε plasmids obtained implied that multiple insertions and deletions had occurred, mediated by different transposons and Class 1 integrons with various ARGs. Our study provides compelling evidence that Class 1 integrons, Tn402-like transposons, Tn3-like transposons and/or IS26 played important roles in the acquisition of ARGs across all investigated plasmids. Our plasmid sequencing data provide new insights into how these mobile genetic elements could mediate the acquisition and spread of ARGs in environmental bacteria.

Hitherto-Unnoticed Self-Transmissible Plasmids Widely Distributed among Different Environments in Japan.

Various conjugative plasmids were obtained by exogenous plasmid capture, biparental mating, and/or triparental mating methods from different environmental samples in Japan. Based on phylogenetic analyses of their whole-nucleotide sequences, new IncP/P-1 plasmids that could be classified into novel subgroups were obtained. Mini-replicons of the plasmids were constructed, and each of them was incompatible with at least one of the IncP/P-1 plasmids, although they showed diverse iteron sequences in their oriV regions. There were two large clades of IncP/P-1 plasmids, clade I and II. Plasmids in clade I and II included antibiotic resistance genes. Notably, nucleotide compositions of newly found plasmids exhibited different tendencies compared with those of the previously well-studied IncP/P-1 plasmids. Indeed, the host range of plasmids of clade II was different from that of clade I. Although few PromA plasmids have been reported, the number of plasmids belonging to PromAß, and -γ subgroups detected in this study was close to that of IncP/P-1 plasmids. The host ranges of PromAγ and PromAδ plasmids were broad and transferred to different and distinct classes of Proteobacteria. Interestingly, PromA plasmids and many IncP/P-1 plasmids do not carry any accessory genes. These findings indicate the presence of "hitherto-unnoticed" conjugative plasmids, including IncP/P-1 or PromA derivative ones in nature. These plasmids would have important roles in the exchange of various genes, including antibiotic resistance genes, among different bacteria in nature. IMPORTANCE Plasmids are known to spread among different bacteria. However, which plasmids spread among environmental samples and in which environments they are present is still poorly understood. This study showed that unidentified conjugative plasmids were present in various environments. Different novel IncP/P-1 plasmids were found, whose host ranges were different from those of known plasmids, showing wide diversity of IncP/P-1 plasmids. PromA plasmids, exhibiting a broad host range, were diversified into several subgroups and widely distributed in varied environments. These findings are important for understanding how bacteria naturally exchange their genes, including antibiotic resistance genes, a growing threat to human health worldwide.

Oxygen concentration affects frequency and range of transconjugants for the incompatibility (Inc) P-1 and P-7 plasmids pBP136 and pCAR1.

The frequency of transconjugants were compared for the incompatibility (Inc) P-1 and P-7 plasmids pBP136 and pCAR1 under aerobic and anaerobic conditions. Filter mating assays were performed with one donor strain and one recipient strain using different donors of Pseudomonas and recipient strains, including Pseudomonas, Pantoea, and Buttiauxella. Under anaerobic condition, frequencies of transconjugants for both plasmids were 101-103-fold lower than those under aerobic condition regardless of whether aerobically or anaerobically grown donors and recipients were used. To compare the transconjugant ranges under aerobic and anaerobic conditions, conjugation was performed between the donor of pBP136 and recipient bacteria extracted from environmental samples. Several transconjugants were uniquely obtained from each aerobic or anaerobic condition. Our findings indicate that a plasmid can differently spread among bacteria depending on the oxygen concentrations of the environment.

Fluviispira sanaruensis sp., nov., Isolated from a Brackish Lake in Hamamatsu, Japan.

Strain RF1110005T, which was isolated from brackish lake water sampled at Lake Sanaru in Japan as a "filterable" bacterial strain, was characterized as a novel species in the genus Fluviispira, family Silvanigrellaceae, order Silvanigrellales, the class Oligoflexia and the phylum Bdellovibrionota. Cells of RF1110005T were aerobic, Gram stain negative, and show a pleomorphic morphology of spiral, filamentous and rod shapes. Catalase reaction was positive. Strain RF1110005T grew optimally at 30 °C, pH 7.0-8.0 and 0.5% NaCl (w/v). The major polar lipids in RF1110005T were phosphatidylethanolamine and phosphatidylglycerol. The predominant cellular fatty acids were iso-C15:0 and anteiso-C15:0. Phylogenetic analysis based on 16S rRNA gene sequences and concatenates of core gene sequence showed that the nearest neighbor of strain RF1110005T was Fluviispira multicolorata strain 33A1-SZDPT with 98.4% 16S rRNA gene sequence similarity. The genome size of strain RF1110005T was 3.5 Mbp with two plasmids (80 kb and 69 kb), and the G + C content was 33.7 mol%. Comparisons with genome-wide analyses and chemotaxonomic characters clearly showed that strain RF1110005T differed from F. multicolorata. Therefore, a novel species in Fluviispira sanaruensis, sp. nov., is proposed for strain RF1110005T (= JCM 31447 T = LMG 30360 T).

Light Response of Pseudomonas putida KT2440 Mediated by Class II LitR, a Photosensor Homolog.

Pseudomonas putida KT2440 retains three homologs (PplR1 to PplR3) of the LitR/CarH family, an adenosyl B12-dependent light-sensitive MerR family transcriptional regulator. Transcriptome analysis revealed the existence of a number of photoinducible genes, including pplR1, phrB (encoding DNA photolyase), ufaM (furan-containing fatty acid synthase), folE (GTP cyclohydrolase I), cryB (cryptochrome-like protein), and multiple genes without annotated/known function. Transcriptional analysis by quantitative reverse transcription-PCR with knockout mutants of pplR1 to pplR3 showed that a triple knockout completely abolished the light-inducible transcription in P. putida, which indicates the occurrence of ternary regulation of PplR proteins. A DNase I footprint assay showed that PplR1 protein specifically binds to the promoter regions of light-inducible genes, suggesting a consensus PplR1-binding direct repeat, 5'-T(G/A)TACAN12TGTA(C/T)A-3'. The disruption of B12 biosynthesis cluster did not affect the light-inducible transcription; however, disruption of ppSB1-LOV (where LOV indicates "light, oxygen, or voltage") and ppSB2-LOV, encoding blue light photoreceptors adjacently located to pplR3 and pplR2, respectively, led to the complete loss of light-inducible transcription. Overall, the results suggest that the three PplRs and two PpSB-LOVs cooperatively regulate the light-inducible gene expression. The wide distribution of the pplR/ppSB-LOV cognate pair homologs in Pseudomonas spp. and related bacteria suggests that the response and adaptation to light are similarly regulated in the group of nonphototrophic bacteria.IMPORTANCE The LitR/CarH family is a new group of photosensor homologous to MerR-type transcriptional regulators. Proteins of this family are distributed to various nonphototrophic bacteria and grouped into at least five classes (I to V). Pseudomonas putida retaining three class II LitR proteins exhibited a genome-wide response to light. All three paralogs were functional and mediated photodependent activation of promoters directing the transcription of light-induced genes or operons. Two LOV (light, oxygen, or voltage) domain proteins, adjacently encoded by two litR genes, were also essential for the photodependent transcriptional control. Despite the difference in light-sensing mechanisms, the DNA binding consensus of class II LitR [T(G/A)TA(C/T)A] was the same as that of class I. This is the first study showing the actual involvement of class II LitR in light-induced transcription.

Multilamellar and Multivesicular Outer Membrane Vesicles Produced by a Buttiauxella agrestis tolB Mutant.

Outer membrane vesicles (OMVs) are naturally released from Gram-negative bacteria and play important roles in various biological functions. Released vesicles are not uniform in shape, size, or characteristics, and little is known about this diversity of OMVs. Here, we show that deletion of tolB, which encodes a part of the Tol-Pal system, leads to the production of multiple types of vesicles and increases overall vesicle production in the high-vesicle-forming Buttiauxella agrestis type strain JCM 1090. The ΔtolB mutant produced small OMVs and multilamellar/multivesicular OMVs (M-OMVs) as well as vesicles with a striking similarity to the wild type. M-OMVs, previously undescribed, contained triple-lamellar membrane vesicles and multiple vesicle-incorporating vesicles. Ultracentrifugation enabled the separation and purification of each type of OMV released from the ΔtolB mutant, and visualization by quick-freeze deep-etch and replica electron microscopy indicated that M-OMVs are composed of several lamellar membranes. Visualization of intracellular compartments of ΔtolB mutant cells showed that vesicles were accumulated in the broad periplasm, which is probably due to the low linkage between the outer and inner membranes attributed to the Tol-Pal defect. The outer membrane was invaginating inward by wrapping a vesicle, and the precursor of M-OMVs existed in the cell. Thus, we demonstrated a novel type of bacterial OMV and showed that unconventional processes enable the B. agrestis ΔtolB mutant to form unique vesicles.IMPORTANCE Membrane vesicle (MV) formation has been recognized as a common mechanism in prokaryotes, and MVs play critical roles in intercellular interaction. However, a broad range of MV types and their multiple production processes make it difficult to gain a comprehensive understanding of MVs. In this work, using vesicle separation and electron microscopic analyses, we demonstrated that diverse types of outer membrane vesicles (OMVs) were released from an engineered strain, Buttiauxella agrestis JCM 1090T ΔtolB mutant. We also discovered a previously undiscovered type of vesicle, multilamellar/multivesicular outer membrane vesicles (M-OMVs), which were released by this mutant using unconventional processes. These findings have facilitated considerable progress in understanding MV diversity and expanding the utility of MVs in biotechnological applications.

Identification of a transcriptional regulator for oligotrophy-responsive promoter in Rhodococcus erythropolis N9T-4.

Two genes, aldA, and mnoA, encoding an NAD-dependent aliphatic dehydrogenase and N,N'-dimethyl-4-nitrosoaniline-dependent methanol dehydrogenase, respectively, are strongly expressed when Rhodococcus erythropolis N9T-4 is grown under oligotrophic conditions. In this study, we found a transcriptional regulator required for the transcription of both aldA and mnoA. The transcriptional regulator was also found to be essential for the oligotrophic growth of N9T-4.

Chryseotalea sanaruensis gen. nov., sp., nov., a Member of the Family Cytophagaceae, Isolated from a Brackish Lake in Hamamatsu Japan.

A strain-designated YsT was isolated as a filterable bacterial strain from Lake Sanaru, a brackish water lake in Hamamatsu Japan. YsT is aerobic, Gram-negative, and slender rod shaped. YsT grew optimally at 30 °C, pH 7.0-8.0 and without the addition of NaCl. MK-7 was the sole isoprenoid quinone. The main cellular polar lipids were phosphatidylethanolamine and unidentified amino- and polar-lipids. The predominant cellular fatty acids were C18:0, iso-C14:0 and iso-C15:0. Phylogenetic analysis of 16S rRNA gene sequence revealed the nearest neighbours of strain YsT to be members of the Ohtaekwangia and Chryseolinea genera with 91.2-92.1% sequence similarity. The percentages of conserved proteins (POCP) between the genomes of YsT and related strains were less than 50%. Phenotypic analyses suggested that YsT could not metabolize glucose and related sugars, which was discriminative from its phylogenetic relatives. We, therefore, propose a novel species in a new genus, Chryseotalea sanaruensis gen. nov., sp. nov. in the family Cytophagaceae (= JCM 30318T = LMG 30359T), based on cell size, the predominant cellular fatty acid composition, and the DNA GC content (38.9 mol%).

Algoriphagus sanaruensis sp. nov., a member of the family Cyclobacteriaceae, isolated from a brackish lake in Hamamatsu, Japan.

Strain M8-2T, which was isolated from brackish lake water (Lake Sanaru) in Japan, was characterized for representation of a novel species in the genus Algoriphagus. Cells of strain M8-2T were aerobic, Gram-stain-negative and curved-rod-shaped (0.2-0.5 µm wide and 0.7-1.9 µm long). Strain M8-2T grew optimally at 30 °C, pH 6.5-7.5 and in the presence of 0.5-1.0 % (w/v) NaCl. MK-7 was the sole isoprenoid quinone. The major polar lipids were phosphatidylethanolamine, an unidentified phospholipid and an unidentified polar lipid. The predominant cellular fatty acids were iso-C15 : 0 and anteiso-C15 : 0. Phylogenetic analysis based on its 16S rRNA gene sequence showed that strain M8-2T belonged to the genus Algoriphagus and was closely related to Algoriphagus aquatilis A8-7T, Algoriphagus boseongensis BS-R1T, Algoriphagus aquaeductus T4T, Algoriphagus olei CC-Hsuan-617T, Algoriphagusshivajiensis NIO-S3T and Algoriphagus mannitolivorans DSM 15301T with sequence similarities of 96.6-97.4 %. Results of average nucleotide identity (<75 %) and digital DNA-DNA hybridization (<19 %) studies showed that M8-2T was distinct from its phylogenetic relatives. Based on the results of tests for acid production, the predominant cellular fatty acid composition, the DNA G+C content and phylogenetic position, a novel species in the genus Algoriphagus, with the name Algoriphagussanaruensis sp. nov., is proposed for strain M8-2T (=JCM 31446T=LMG 29969T).

Divalent cations increase the conjugation efficiency of the incompatibility P-7 group plasmid pCAR1 among different Pseudomonas hosts.

The incompatibility (Inc) P-7 group plasmid pCAR1 can be efficiently transferred among bacteria in artificial microcosms in the presence of divalent cations Ca2+ and Mg2+. One-on-one mating assays between Pseudomonas strains with different plasmids showed that the promotion of conjugation efficiency by divalent cations was exhibited in other plasmids, including pB10 and NAH7; however, this effect was larger in IncP-7 plasmids. The impact on pCAR1 conjugation differed according to donor-recipient pairs, and conjugation efficiency promotion was clearly detected between the donors P. resinovorans CA10dm4 and P. fluorescens Pf0-1 and the recipients P. putida KT2440 and CA10dm4. Transcriptome analyses showed that pCAR1 gene expression did not respond to cation changes, including the tra/trh genes involved in its transfer. However, the transcription of oprH genes, encoding putative outer-membrane proteins in both the donor and the recipient, were commonly upregulated under cation-limited conditions. The conjugation frequency of pCAR1 in the KT2440 oprH mutant was found not to respond to cations. This effect was partially recovered by complementation with the oprH gene, suggesting that OprH is involved in the increase of pCAR1 conjugation efficiency by divalent cations.

Proteobacteria and Bacteroidetes are major phyla of filterable bacteria passing through 0.22 µm pore size membrane filter, in Lake Sanaru, Hamamatsu, Japan.

141 filterable bacteria that passed through a 0.22 µm pore size filter were isolated from Lake Sanaru in Hamamatsu, Japan. These belonged to Proteobacteria, Bacteroidetes, Firmicutes, or Actinobacteria among which the first two phyla comprised the majority of the isolates. 48 isolates (12 taxa) are candidates assignable to new bacterial species or genera of Proteobacteria or Bacteroidetes.

The behavior of mobile genetic elements (MGEs) in different environments.

Mobile genetic elements (MGEs) including plasmids have an important role in the rapid evolution and adaptation of bacteria. Here, the behavior of MGEs in different environments is reviewed, in particular, behavior of the plasmid pCAR1, a carbazole-degradative plasmid isolated from Pseudomonas resinovorans CA10. pCAR1 belongs to incompatibility P-7 group and is self-transmissible among different bacteria. Comparisons of changes in the transcriptome of different host strains caused by carrying pCAR1 revealed common responses in the hosts and host-specific responses. Monitoring the survival of the host and transfer of the plasmid in artificial and natural environmental samples revealed several environmental factors, including cations and water content, which changed the behavior of both the host and its plasmid. Single-cell level analysis to detect the transconjugants of different plasmids successfully determined the transfer range of the plasmids. Three nucleoid-associated proteins encoded on pCAR1 are important factors affecting its genetic stability, maintenance, and transfer.

MvaT Family Proteins Encoded on IncP-7 Plasmid pCAR1 and the Host Chromosome Regulate the Host Transcriptome Cooperatively but Differently.

MvaT proteins are members of the H-NS family of proteins in pseudomonads. The IncP-7 conjugative plasmid pCAR1 carries an mvaT-homologous gene, pmr. In Pseudomonas putida KT2440 bearing pCAR1, pmr and the chromosomally carried homologous genes, turA and turB, are transcribed at high levels, and Pmr interacts with TurA and TurB in vitro. In the present study, we clarified how the three MvaT proteins regulate the transcriptome of P. putida KT2440(pCAR1). Analyses performed by a modified chromatin immunoprecipitation assay with microarray technology (ChIP-chip) suggested that the binding regions of Pmr, TurA, and TurB in the P. putida KT2440(pCAR1) genome are almost identical; nevertheless, transcriptomic analyses using mutants with deletions of the genes encoding the MvaT proteins during the log and early stationary growth phases clearly suggested that their regulons were different. Indeed, significant regulon dissimilarity was found between Pmr and the other two proteins. Transcription of a larger number of genes was affected by Pmr deletion during early stationary phase than during log phase, suggesting that Pmr ameliorates the effects of pCAR1 on host fitness more effectively during the early stationary phase. Alternatively, the similarity of the TurA and TurB regulons implied that they might play complementary roles as global transcriptional regulators in response to plasmid carriage.

Comparisons of the transferability of plasmids pCAR1, pB10, R388, and NAH7 among Pseudomonas putida at different cell densities.

The transferability of plasmids pCAR1, pB10, R388, and NAH7 was compared using the same donor-recipient system at different cell density combinations in liquid or on a solid surface. pCAR1 was efficiently transferred in liquid, whereas the other plasmids were preferentially transferred on a solid surface. Difference of liquid or solid affected the transfer frequency especially at lower cell densities.

Dominant ectosymbiotic bacteria of cellulolytic protists in the termite gut also have the potential to digest lignocellulose.

Wood-feeding lower termites harbour symbiotic gut protists that support the termite nutritionally by degrading recalcitrant lignocellulose. These protists themselves host specific endo- and ectosymbiotic bacteria, functions of which remain largely unknown. Here, we present draft genomes of a dominant, uncultured ectosymbiont belonging to the order Bacteroidales, 'Candidatus Symbiothrix dinenymphae', which colonizes the cell surface of the cellulolytic gut protists Dinenympha spp. We analysed four single-cell genomes of Ca. S. dinenymphae, the highest genome completeness was estimated to be 81.6-82.3% with a predicted genome size of 4.28-4.31 Mb. The genome retains genes encoding large parts of the amino acid, cofactor and nucleotide biosynthetic pathways. In addition, the genome contains genes encoding various glycoside hydrolases such as endoglucanases and hemicellulases. The genome indicates that Ca. S. dinenymphae ferments lignocellulose-derived monosaccharides to acetate, a major carbon and energy source of the host termite. We suggest that the ectosymbiont digests lignocellulose and provides nutrients to the host termites, and hypothesize that the hydrolytic activity might also function as a pretreatment for the host protist to effectively decompose the crystalline cellulose components.

Modulation of primary cell function of host Pseudomonas bacteria by the conjugative plasmid pCAR1.

The impacts of plasmid carriage on the host cell were comprehensively analysed using the conjugative plasmid pCAR1 in three different Pseudomonas hosts, P. putida KT2440, P. aeruginosa PAO1 and P. fluorescens Pf0-1. Plasmid carriage reduced host fitness, swimming motility, and resistance to osmotic or pH stress. Plasmid carriage brought about alterations in primary metabolic capacities in the TCA cycle of the hosts. Differentially transcribed genes in the three hosts associated with plasmid carriage were identified by growth phase-dependent transcriptome analyses. Plasmid carriage commonly showed a greater effect on the host transcriptome at the transition and early stationary phases. The transcriptome alterations were similar between KT2440 and PAO1. Transcriptions of numbers of genes encoding ribosomal proteins, F-type ATPase, and RNAP core in both strains were not suppressed enough in the early stationary phase by plasmid carriage. These responses may have been responsible for the reduction in host fitness, motility and stress resistances. Host-specific responses to plasmid carriage were transcriptional changes of genes on putative prophage or foreign DNA regions. The extents of the impacts on host phenotypes and transcriptomes were similarly greatest in KT2440 and lowest in Pf0-1. These findings suggest that host cell function was actively regulated by plasmid carriage.

Effects of three different nucleoid-associated proteins encoded on IncP-7 plasmid pCAR1 on host Pseudomonas putida KT2440.

Nucleoid-associated proteins (NAPs), which fold bacterial DNA and influence gene transcription, are considered to be global transcriptional regulators of genes on both plasmids and the host chromosome. Incompatibility P-7 group plasmid pCAR1 carries genes encoding three NAPs: H-NS family protein Pmr, NdpA-like protein Pnd, and HU-like protein Phu. In this study, the effects of single or double disruption of pmr, pnd, and phu were assessed in host Pseudomonas putida KT2440. When pmr and pnd or pmr and phu were simultaneously disrupted, both the segregational stability and the structural stability of pCAR1 were markedly decreased, suggesting that Pmr, Pnd, and Phu act as plasmid-stabilizing factors in addition to their established roles in replication and partition systems. The transfer frequency of pCAR1 was significantly decreased in these double mutants. The segregational and structural instability of pCAR1 in the double mutants was recovered by complementation of pmr, whereas no recovery of transfer deficiency was observed. Comprehensive phenotype comparisons showed that the host metabolism of carbon compounds, which was reduced by pCAR1 carriage, was restored by disruption of the NAP gene(s). Transcriptome analyses of mutants indicated that transcription of genes for energy production, conversion, inorganic ion transport, and metabolism were commonly affected; however, how their products altered the phenotypes of mutants was not clear. The findings of this study indicated that Pmr, Pnd, and Phu act synergistically to affect pCAR1 replication, maintenance, and transfer, as well as to alter the host metabolic phenotype.

Nucleoid-associated proteins encoded on plasmids: Occurrence and mode of function.

Nucleoid-associated proteins (NAPs) play a role in changing the shape of microbial DNA, making it more compact and affecting the regulation of transcriptional networks in host cells. Genes that encode NAPs include H-NS family proteins (H-NS, Ler, MvaT, BpH3, Bv3F, HvrA, and Lsr2), FIS, HU, IHF, Lrp, and NdpA, and are found in both microbial chromosomes and plasmid DNA. In the present study, NAP genes were distributed among 442 plasmids out of 4602 plasmid sequences, and many H-NS family proteins, and HU, IHF, Lrp, and NdpA were found in plasmids of Alpha-, Beta-, and Gammaproteobacteria, while HvrA, Lsr2, HU, and Lrp were found in other classes including Actinobacteria and Bacilli. Larger plasmids frequently carried multiple NAP genes. In addition, NAP genes were more frequently found in conjugative plasmids than non-transmissible plasmids. Several host cells carried the same types of H-NS family proteins on both their plasmids and chromosome(s), while this was not observed for other NAPs. Recent studies have shown that NAP genes on plasmids and chromosomes play important roles in the physical and regulatory integration of plasmids into the host cell.