Denitrification in low oxic environments increases the accumulation of nitrogen oxide intermediates and modulates the evolutionary potential of microbial populations.

Denitrification in oxic environments occurs when a microorganism uses nitrogen oxides as terminal electron acceptors even though oxygen is available. While this phenomenon is well-established, its consequences on ecological and evolutionary processes remain poorly understood. We hypothesize here that denitrification in oxic environments can modify the accumulation profiles of nitrogen oxide intermediates with cascading effects on the evolutionary potentials of denitrifying microorganisms. To test this, we performed laboratory experiments with Paracoccus denitrificans and complemented them with individual-based computational modelling. We found that denitrification in low oxic environments significantly increases the accumulation of nitrite and nitric oxide. We further found that the increased accumulation of these intermediates has a negative effect on growth at low pH. Finally, we found that the increased negative effect at low pH increases the number of individuals that contribute to surface-associated growth. This increases the amount of genetic diversity that is preserved from the initial population, thus increasing the number of genetic targets for natural selection to act upon and resulting in higher evolutionary potentials. Together, our data highlight that denitrification in low oxic environments can affect the ecological processes and evolutionary potentials of denitrifying microorganisms by modifying the accumulation of nitrogen oxide intermediates.

Identification and cultivation of anaerobic bacterial scavengers of dead cells.

The cycle of life and death and Earth's carbon cycle(s) are intimately linked, yet how bacterial cells, one of the largest pools of biomass on Earth, are recycled back into the carbon cycle remains enigmatic. In particular, no bacteria capable of scavenging dead cells in oxygen-depleted environments have been reported thus far. In this study, we discover the first anaerobes that scavenge dead cells and the two isolated strains use distinct strategies. Based on live-cell imaging, transmission electron microscopy, and hydrolytic enzyme assays, one strain (designated CYCD) relied on cell-to-cell contact and cell invagination for degrading dead food bacteria where as the other strain (MGCD) degraded dead food bacteria via excretion of lytic extracellular enzymes. Both strains could degrade dead cells of differing taxonomy (bacteria and archaea) and differing extents of cell damage, including those without artificially inflicted physical damage. In addition, both depended on symbiotic metabolic interactions for maximizing cell degradation, representing the first cultured syntrophic Bacteroidota. We collectively revealed multiple symbiotic bacterial decomposition routes of dead prokaryotic cells, providing novel insight into the last step of the carbon cycle.

Complete Genome Sequence of Dyella sp. Strain GSA-30, a Predominant Endophytic Bacterium of Dendrobium Plants.

We report a complete genome sequence of Dyella sp. strain GSA-30, a predominant endophytic bacterium of Dendrobium plants. The genome consists of a circular 5,501,810-bp chromosome with a G+C content of 61.4%. The genome was predicted to harbor 6 rRNA genes, 51 tRNA genes, and 4,713 coding sequences.

Complete Genome Sequence of Flavobacterium sp. Strain GSB-24, Isolated from inside Dendrobium Roots.

Here, we report a complete genome sequence of Flavobacterium sp. strain GSB-24, an endophytic bacterium of Dendrobium plants. The genome consists of a circular 5,286,830-bp chromosome with a G+C content of 33.8% and a circular 64,374-bp plasmid with a G+C content of 29.3%.

Granulimonas faecalis gen. nov., sp. nov., and Leptogranulimonas caecicola gen. nov., sp. nov., novel lactate-producing Atopobiaceae bacteria isolated from mouse intestines, and an emended description of the family Atopobiaceae.

Two strictly anaerobic, Gram-stain-positive, non-motile bacteria (strains OPF53T and TOC12T) were isolated from mouse intestines. Strains OPF53T and TOC12T grew at pH 5.5-9.0 and 5.0-9.0, respectively, and at temperatures of 30-45 °C. The cell morphologies of these strains were short rods and rods, respectively, and the cells possessed intracellular granules. The major cellular fatty acids of OPF53T were C18  :  1 cis 9 and C18  :  1 cis 9 dimethyl acetal, whereas those of TOC12T were C18  :  0 and C18  :  1 cis 9. In OPF53T, the main end-products of modified peptone-yeast extract-glucose (PYG) fermentation were lactate, formate and butyrate, whereas, in addition to these acids, TOC12T also produced hydrogen. The genomes of OPF53T and TOC12T were respectively 2.2 and 2.0 Mbp in size with a DNA G+C contents of 69.1 and 58.7 %. The 16S rRNA gene sequences of OPF53T and TOC12T showed the highest similarity to members of the family Atopobiaceae, namely, Olsenella phocaeensis Marseille-P2936T (94.3 %) and Olsenella umbonata KCTC 15140T (93.2 %), respectively. Phylogenetic analyses revealed that both isolates formed distinct lineages from other genera of the family Atopobiaceae. In addition, the two strains were characterized by relatively low 16S rRNA gene sequence similarity (93.4 %) and can be distinguished by their distinctive traits (including cell shape, DNA G+C content, and major fatty acids profiles). On the basis of their polyphasic taxonomic properties, these isolates represent two noel species of two novel genera within the family Atopobiaceae, for which the names Granulimonas faecalis gen. nov., sp. nov. (OPF53T=JCM 35015T=KCTC 25474T) and Leptogranulimonas caecicola gen. nov., sp. nov. (TOC12T=JCM 35017T=KCTC 25472T) are proposed.

Bile Salt Hydrolases with Extended Substrate Specificity Confer a High Level of Resistance to Bile Toxicity on Atopobiaceae Bacteria.

The bile resistance of intestinal bacteria is among the key factors responsible for their successful colonization of and survival in the mammalian gastrointestinal tract. In this study, we demonstrated that lactate-producing Atopobiaceae bacteria (Leptogranulimonas caecicola TOC12T and Granulimonas faecalis OPF53T) isolated from mouse intestine showed high resistance to mammalian bile extracts, due to significant bile salt hydrolase (BSH) activity. We further succeeded in isolating BSH proteins (designated LcBSH and GfBSH) from L. caecicola TOC12T and G. faecalis OPF53T, respectively, and characterized their enzymatic features. Interestingly, recombinant LcBSH and GfBSH proteins exhibited BSH activity against 12 conjugated bile salts, indicating that LcBSH and GfBSH have much broader substrate specificity than the previously identified BSHs from lactic acid bacteria, which are generally known to hydrolyze six bile salt isomers. Phylogenetic analysis showed that LcBSH and GfBSH had no affinities with any known BSH subgroup and constituted a new BSH subgroup in the phylogeny. In summary, we discovered functional BSHs with broad substrate specificity from Atopobiaceae bacteria and demonstrated that these BSH enzymes confer bile resistance to L. caecicola TOC12T and G. faecalis OPF53T.

Phage Genes Induce Quorum Sensing Signal Release through Membrane Vesicle Formation.

Membrane vesicles (MVs) released from the bacterium Paracoccus denitrificans Pd1222 are enriched with the quorum sensing (QS) signaling molecule N-hexadecanoyl-l-homoserine lactone (C16-HSL). However, the biogenesis of MVs in Pd1222 remains unclear. Investigations on MV formation are crucial for obtaining a more detailed understanding of the dynamics of MV-assisted signaling. In the present study, live-cell imaging showed that P. denitrificans Pd1222 produced MVs through cell lysis under DNA-damaging conditions. DNA sequencing of MVs and a transcriptome ana-lysis of cells indicated that the expression of a prophage region was up-regulated at the onset of MV formation under DNA-damaging conditions. A further sequence ana-lysis identified a putative endolysin (Pden_0381) and holin (Pden_0382) in the prophage region. The expression of these genes was regulated by RecA. Using gene knockout mutants, we showed that prophage-encoded endolysin was critical for MV formation by P. denitrificans Pd1222 under DNA-damaging conditions. MV triggering by endolysin was dependent on the putative holin, which presumably transported endolysin to the periplasmic space. C16-HSL quantification revealed that more signals were released into the milieu as a consequence of the effects of endolysin. Using a QS reporter strain, we found that the QS response in P. denitrificans was stimulated by inducing the expression of endolysin. Collectively, these results provide novel insights into the mechanisms by which a bacterial cell-to-cell communication system is manipulated by phage genes.

Complete Genome Sequence of Lactobacillus helveticus JCM 1004, an Aminopeptidase-Producing Lactic Acid Bacterium.

We report the complete genome sequence of Lactobacillus helveticus JCM 1004, an aminopeptidase-producing lactic acid bacterium. The genome consists of a circular chromosome which comprises 2,261,280 bp, with a G+C content of 37.56%. The genome was predicted to harbor 13 rRNA genes, 64 tRNA genes, and 2,462 protein-coding sequences.

Complete Genome Sequence of Atopobiaceae Bacterium Strain P1, Isolated from Mouse Feces.

Atopobiaceae bacterium strain P1 (Actinobacteria, Coriobacteriales) was isolated from mouse feces. Here, we report the complete genome sequence of this strain, which has a total size of 2,028,478 bp and a G+C content of 58.6%.

Identification of Bile Salt Hydrolase and Bile Salt Resistance in a Probiotic Bacterium Lactobacillus gasseri JCM1131T.

Lactobacillus gasseri is one of the most likely probiotic candidates among many Lactobacillus species. Although bile salt resistance has been defined as an important criterion for selection of probiotic candidates since it allows probiotic bacteria to survive in the gut, both its capability and its related enzyme, bile salt hydrolase (BSH), in L. gasseri is still largely unknown. Here, we report that the well-known probiotic bacterium L. gasseri JCM1131T possesses BSH activity and bile salt resistance capability. Indeed, this strain apparently showed BSH activity on the plate assay and highly tolerated the primary bile salts and even taurine-conjugated secondary bile salt. We further isolated a putative BSH enzyme (LagBSH) from strain JCM1131T and characterized the enzymatic function. The purified LagBSH protein exhibited quite high deconjugation activity for taurocholic acid and taurochenodeoxycholic acid. The lagBSH gene was constitutively expressed in strain JCM1131T, suggesting that LagBSH likely contributes to bile salt resistance of the strain and may be associated with survival capability of strain JCM1131T within the human intestine by bile detoxification. Thus, this study first demonstrated the bile salt resistance and its responsible enzyme (BSH) activity in strain JCM1131T, which further supports the importance of the typical lactic acid bacterium as probiotics.

Complete Genome Sequence of Anaerostipes caccae Strain L1-92T, a Butyrate-Producing Bacterium Isolated from Human Feces.

Anaerostipes caccae strain L1-92T is a well-known butyrate-producing bacterium that has been isolated from human feces. In this announcement, we present the complete genome sequence of A. caccae strain L1-92T, which comprises 3,590,719 bp with a G+C content of 44.30%. The genome harbors 3,369 predicted protein-coding genes.

Involvement of membrane vesicles in long-chain-AHL delivery in Paracoccus species.

Bacteria are known to communicate with each other through signalling molecules that regulate gene expression within the population. However, the way in which hydrophobic signals are released and transmitted among bacterial population is not well understood. Recent studies show that membrane vesicles (MVs) are involved in delivering hydrophobic signals, such as in N-hexadecanoyl-l-homoserine lactone (C16-HSL) signalling in Paracoccus denitrificans Pd1222. In this study, we identified the AHLs produced in Paracoccus aminophilus JCM7686, Paracoccus aminovorans NBRC16711, Paracoccus thiocyanatus JCM20756, Paracoccus versutus JCM20754 and Paracoccus yeei ATCC BAA-599, and show that the main AHL produced in all the strains is C16-HSL. Our results show that these Paracoccus species also release MVs that carry C16-HSL, but at different proportions. Most of the strains carry C16-HSL in MVs, while in P. aminophilus JCM7686, very little C16-HSL was detected in MVs, but was found in other fractions of the supernatant. Given the utilization of a common signal, we showed that these Paracoccus species can share signals with P. denitrificans Pd1222, and examined the role of MVs in signalling. Our study provides new insights into the way in which bacteria communicate using hydrophobic signals.

Peculiarities of biofilm formation by Paracoccus denitrificans.

Most bacteria form biofilms, which are thick multicellular communities covered in extracellular matrix. Biofilms can become thick enough to be even observed by the naked eye, and biofilm formation is a tightly regulated process. Paracoccus denitrificans is a non-motile, Gram-negative bacterium that forms a very thin, unique biofilm. A key factor in the biofilm formed by this bacterium is a large surface protein named biofilm-associated protein A (BapA), which was recently reported to be regulated by cyclic diguanosine monophosphate (cyclic-di-GMP or c-di-GMP). Cyclic-di-GMP is a major second messenger involved in biofilm formation in many bacteria. Though cyclic-di-GMP is generally reported as a positive regulatory factor in biofilm formation, it represses biofilm formation in P. denitrificans. Furthermore, quorum sensing (QS) represses biofilm formation in this bacterium, which is also reported as a positive regulator of biofilm formation in most bacteria. The QS signal used in P. denitrificans is hydrophobic and is delivered through membrane vesicles. Studies on QS show that P. denitrificans can potentially form a thick biofilm but maintains a thin biofilm under normal growth conditions. In this review, we discuss the peculiarities of biofilm formation by P. denitrificans with the aim of deepening the overall understanding of bacterial biofilm formation and functions.

Polyfunctional Nanofibril Appendages Mediate Attachment, Filamentation, and Filament Adaptability in Leptothrix cholodnii.

Leptothrix is a species of Fe/Mn-oxidizing bacteria known to form long filaments composed of chains of cells that eventually produce a rigid tube surrounding the filament. Prior to the formation of this brittle microtube, Leptothrix cells secrete hair-like structures from the cell surface, called nanofibrils, which develop into a soft sheath that surrounds the filament. To clarify the role of nanofibrils in filament formation in L. cholodnii SP-6, we analyze the behavior of individual cells and multicellular filaments in high-aspect ratio microfluidic chambers using time-lapse and intermittent in situ fluorescent staining of nanofibrils, complemented with atmospheric scanning electron microscopy. We show that in SP-6 nanofibrils are important for attachment and their distribution on young filaments post-attachment is correlated to the directionality of filament elongation. Elongating filaments demonstrate a surprising ability to adapt to their physical environment by changing direction when they encounter obstacles: they bend or reverse direction depending on the angle of the collision. We show that the forces involved in the collision can be used to predict the behavior of filament. Finally, we show that as filaments grow in length, the older region becomes confined by the sheath, while the newly secreted nanofibrils at the leading edge of the filament form a loose, divergent, structure from which cells periodically escape.

Paracoccus denitrificans can utilize various long-chain N-acyl homoserine lactones and sequester them in membrane vesicles.

Many gram-negative bacteria utilize N-acyl homoserine lactone (AHL) signals to communicate with each other. Once they have been released, these signals are assumed to be shared among the population in the local environment. In contrast to this canonical quorum-sensing (QS) model, recent study in Paracoccus denitrificans showed that they can traffic their signals to each other via membrane vesicles (MVs). Here, we demonstrate that various long-chain AHLs inhibited cell aggregation in P. denitrificans, whereas the short-chain AHLs alone did not. Furthermore, MVs released from P. denitrificans were able to take up the long-chain AHLs from the environment into MVs. The AHLs associated with MVs triggered gene expression in P. denitrificans, indicating their role in QS. Our results suggest that P. denitrificans can sequester the AHL produced by other bacteria and deliver the signals to themselves via MVs. Utilizing the signals from other bacteria may be advantageous for P. denitrificans to reach the threshold QS concentration in a polymicrobial community in which the population of its own species is relatively low.

Membrane vesicle-mediated bacterial communication.

The classical quorum-sensing (QS) model is based on the assumption that diffusible signaling molecules accumulate in the culture medium until they reach a critical concentration upon which expression of target genes is triggered. Here we demonstrate that the hydrophobic signal N-hexadecanoyl-L-homoserine lactone, which is produced by Paracoccus sp., is released from cells by the aid of membrane vesicles (MVs). Packed into MVs, the signal is not only solubilized in an aqueous environment but is also delivered with varying propensities to different bacteria. We propose a novel MV-based mechanism for binary trafficking of hydrophobic signal molecules, which may be particularly relevant for bacteria that live in open aqueous environments.

The Pseudomonas Quinolone Signal Inhibits Biofilm Development of Streptococcus mutans.

Bacteria often thrive in natural environments through a sessile mode of growth, known as the biofilm. Biofilms are well-structured communities and their formation is tightly regulated. However, the mechanisms by which interspecies interactions alter the formation of biofilms have not yet been elucidated in detail. We herein demonstrated that a quorum-sensing signal in Pseudomonas aeruginosa (the Pseudomonas quinolone signal; PQS) inhibited biofilm formation by Streptococcus mutans. Although the PQS did not affect cell growth, biofilm formation was markedly inhibited. Our results revealed a unique role for this multifunctional PQS and also indicated its application in the development of prophylactic agents against caries-causing S. mutans.