Ribosomal-processing cysteine protease homolog modulates Streptococcus mutans glucan production and interkingdom interactions.

Glucan-dependent biofilm formation is a crucial process in the establishment of Streptococcus mutans as a cariogenic oral microbe. The process of glucan formation has been investigated in great detail, with glycosyltransferases GtfB, GtfC, and GtfD shown to be indispensable for the synthesis of glucans from sucrose. Glucan production can be visualized during biofilm formation through fluorescent labeling, and its abundance, as well as the effect of glucans on general biofilm architecture, is a common phenotype to study S. mutans virulence regulation. Here, we describe an entirely new phenotype associated with glucan production, caused by a mutation in the open reading frame SMU_848, which is located in an operon encoding ribosome-associated proteins. This mutation led to the excess production and accumulation of glucan-containing droplets on the surface of biofilms formed on agar plates after prolonged incubation. While not characterized in S. mutans, SMU_848 shows homology to the phage-related ribosomal protease Prp, essential in cleaving off the N-terminal extension of ribosomal protein L27 for functional ribosome assembly in Staphylococcus aureus. We present a further characterization of SMU_848/Prp, demonstrating that the deletion of this gene leads to significant changes in S. mutans gtfBC expression. Surprisingly, it also profoundly impacts the interkingdom interaction between S. mutans and Candida albicans, a relevant dual-species interaction implicated in severe early childhood caries. The presented data support a potential broader role for SMU_848/Prp, possibly extending its functionality beyond the ribosomal network to influence important ecological processes. IMPORTANCE: Streptococcus mutans is an important member of the oral biofilm and is implicated in the initiation of caries. One of the main virulence mechanisms is the glucan-dependent formation of biofilms. We identified a new player in the regulation of glucan production, SMU_848, which is part of an operon that also encodes for ribosomal proteins L27 and L21. A mutation in SMU_848, which encodes a phage-related ribosomal protease Prp, leads to a significant accumulation of glucan-containing droplets on S. mutans biofilms, a previously unknown phenotype. Further investigations expanded our knowledge about the role of SMU_848 beyond its role in glucan production, including significant involvement in interkingdom interactions, thus potentially playing a global role in the virulence regulation of S. mutans.

Mass spectrometry and split luciferase complementation assays reveal the MecA protein interactome of Streptococcus mutans.

MecA is a highly conserved adaptor protein encoded by prokaryotes from the Bacillota phylum. MecA mutants exhibit similar pleiotropic defects in a variety of organisms, although most of these phenotypes currently lack a mechanistic basis. MecA mediates ClpCP-dependent proteolysis of its substrates, but only several such substrates have been reported in the literature and there are suggestions that proteolysis-independent regulatory mechanisms may also exist. Here, we provide the first comprehensive characterization of the MecA interactome and further assess its regulatory role in Clp-dependent proteolysis. Untargeted coimmunoprecipitation assays coupled with mass spectrometry revealed that the MecA ortholog from the oral pathobiont Streptococcus mutans likely serves as a major protein interaction network hub by potentially complexing with >100 distinct protein substrates, most of which function in highly conserved metabolic pathways. The interactome results were independently verified using a newly developed prokaryotic split luciferase complementation assay (SLCA) to detect MecA protein-protein interactions in vivo. In addition, we further develop a new application of SLCA to support in vivo measurements of MecA relative protein binding affinities. SLCA results were independently verified using targeted coimmunoprecipitation assays, suggesting the general utility of this approach for prokaryotic protein-protein interaction studies. Our results indicate that MecA indeed regulates its interactome through both Clp-dependent proteolysis as well as through an as-yet undefined proteolysis-independent mechanism that may affect more than half of its protein interactome. This suggests a significant aspect of the MecA regulatory function still has yet to be discovered.IMPORTANCEDespite multiple decades of study, the regulatory mechanism and function of MecA have remained largely a mystery. The current study provides the first detailed roadmap to investigate these functions in other medically significant bacteria. Furthermore, this study developed new genetic approaches to assay prokaryotic protein-protein interactions via the split luciferase complementation assay (SLCA). SLCA technology is commonly employed in eukaryotic genetic research but has not yet been established for studies of bacterial protein-protein interactions. The SLCA protein binding affinity assay described here is a new technological advance exclusive to the current study and has not been reported elsewhere.

Employing Cloning-Independent Mutagenesis of Parvimonas micra for the Study of Cell Wall Biogenesis.

The cell wall plays an important structural role for bacteria and is intimately tied to a variety of critical processes ranging from growth and differentiation to pathogenesis. Our understanding of cell wall biogenesis is primarily derived from a relatively small number of heavily studied model organisms. Consequently, these processes can only be inferred for the vast majority of prokaryotes, especially among groups of uncharacterized and/or genetically intractable organisms. Recently, we developed the first tractable genetic system for Parvimonas micra, which is a ubiquitous Gram-positive pathobiont of the human microbiome involved in numerous types of inflammatory infections as well as a variety of malignant tumors. P. micra is also the first, and currently only, member of the entire Tissierellia class of the Bacillota phylum in which targeted genetic manipulation has been demonstrated. Thus, it is now possible to study cell wall biogenesis mechanisms within a member of the Tissierellia, which may also reveal novel aspects of P. micra pathobiology. Herein, we describe a procedure for cloning-independent genetic manipulation of P. micra, including allelic replacement mutagenesis and genetic complementation. The described techniques are also similarly applicable for the study of other aspects of P. micra pathobiology and physiology.

Adaptation of Prokaryotic Toxins for Negative Selection and Cloning-Independent Markerless Mutagenesis in Streptococcus Species.

The Streptococcus mutans genetic system offers a variety of strategies to rapidly engineer targeted chromosomal mutations. Previously, we reported the first S. mutans negative selection system that functions in a wild-type background. This system utilizes induced sensitivity to the toxic amino acid analog p-chlorophenylalanine (4-CP) as a negative selection mechanism and was developed for counterselection-based cloning-independent markerless mutagenesis (CIMM). While we have employed this system extensively for our ongoing genetic studies, we have encountered a couple limitations with the system, mainly its narrow host range and the requirement for selection on a toxic substrate. Here, we report the development of a new negative selection system that addresses both limitations, while still retaining the utility of the previous 4-CP-based markerless mutagenesis system. We placed a variety of toxin-encoding genes under the control of the xylose-inducible gene expression cassette (Xyl-S) and found the Fst-sm and ParE toxins to be suitable candidates for inducible negative selection. We combined the inducible toxins with an antibiotic resistance gene to create several different counterselection cassettes. The most broadly useful of these contained a wild-type fst-sm open reading frame transcriptionally fused to a point mutant form of the Xyl-S expression system, which we subsequently named IFDC4. IFDC4 was shown to exhibit exceptionally low background resistance, with 3- to 4-log reductions in cell number observed when plating on xylose-supplemented medium. IFDC4 also functioned similarly in multiple strains of S. mutans as well as with Streptococcus gordonii and Streptococcus sanguinis. We performed CIMM with IFDC4 and successfully engineered a variety of different types of markerless mutations in all three species. The counterselection strategy described here provides a template approach that should be adaptable for the creation of similar counterselection systems in many other bacteria. IMPORTANCE Multiple medically significant Streptococcus species, such as S. mutans, have highly sophisticated genetic systems available, largely as a consequence of their amenability to genetic manipulation via natural competence. Despite this, few options are available for the creation of markerless mutations in streptococci, especially within wild-type strains. Markerless mutagenesis is a critical tool for genetic studies, as it allows the user to explore many fundamental questions that are not easily addressable using marked mutagenesis. Here, we describe a new approach for streptococcal markerless mutagenesis that offers a variety of advantages over the current approach, which employs induced sensitivity to the toxic substrate 4-CP. The approach employed here should be readily adaptable for the creation of similar markerless mutagenesis systems in other organisms.

Who is in the driver's seat? Parvimonas micra: An understudied pathobiont at the crossroads of dysbiotic disease and cancer.

Recent advances in our understanding of microbiome composition at sites of inflammatory dysbiosis have triggered a substantial interest in a variety of historically understudied bacteria, especially among fastidious obligate anaerobes. A plethora of new evidence suggests that these microbes play outsized roles in establishing synergistic polymicrobial infections at many different sites in the human body. Parvimonas micra is a prime example of such an organism. Despite being almost completely uncharacterized at the genetic level, it is one of the few species commonly detected in abundance at multiple mucosal sites experiencing either chronic or acute inflammatory diseases, and more recently, it has been proposed as a discriminating biomarker for multiple types of malignancies. In the absence of disease, P. micra is commonly found in low abundance, typically residing within the oral cavity and gastrointestinal tract. P. micra exhibits the typical features of an inflammophilic organism, meaning its growth actually benefits from active inflammation and inflammatory tissue destruction. In this mini-review, we will describe our current understanding of this underappreciated but ubiquitous pathobiont, specifically focusing upon the role of P. micra in polymicrobial inflammatory dysbiosis and cancer as well as the key emerging questions regarding its pathobiology. Through this timely work, we highlight Parvimonas micra as a significant driver of disease and discuss its unique position at the crossroads of dysbiosis and cancer.

Development of the First Tractable Genetic System for Parvimonas micra, a Ubiquitous Pathobiont in Human Dysbiotic Disease.

Parvimonas micra is a Gram-positive obligate anaerobe and a typical member of the human microbiome. P. micra is among the most highly enriched species at numerous sites of mucosal dysbiotic disease and is closely associated with the development of multiple types of malignant tumors. Despite its strong association with disease, surprisingly little is known about P. micra pathobiology, which is directly attributable to its longstanding genetic intractability. To address this problem, we directly isolated a collection of P. micra strains from odontogenic abscess clinical specimens and then screened these isolates for natural competence. Amazingly, all of the P. micra clinical isolates exhibited various levels of natural competence, including the reference strain ATCC 33270. By exploiting this ability, we were able to employ cloning-independent methodologies to engineer and complement a variety of targeted chromosomal genetic mutations directly within low-passage-number clinical isolates. To develop a tractable genetic system for P. micra, we first adapted renilla-based bioluminescence for highly sensitive reporter studies. This reporter system was then applied for the development of the novel Theo+ theophylline-inducible riboswitch for tunable gene expression studies over a broad dynamic range. Finally, we demonstrate the feasibility of generating mariner-based transposon sequencing (Tn-seq) libraries for forward genetic screening in P. micra. With the availability of a highly efficient transformation protocol and the current suite of genetic tools, P. micra should now be considered a fully genetically tractable organism suitable for molecular genetic research. The methods presented here provide a clear path to investigate the understudied role of P. micra in polymicrobial infections and tumorigenesis. IMPORTANCE Parvimonas micra is among the most highly enriched species at numerous sites of mucosal dysbiotic disease and is closely associated with numerous cancers. Despite this, little is known about P. micra pathobiology, which is directly attributable to its longstanding genetic intractability. In this study, we provide the first report of P. micra natural competence and describe the only tractable genetic system for this species. The methods presented here will allow for the detailed study of P. micra and its role in infection and tumorigenesis.

The transcription regulator BrsR serves as a network hub of natural competence protein-protein interactions in Streptococcus mutans.

Genome evolution is an essential and stringently regulated aspect of biological fitness. For bacteria, natural competence is one of the principal mechanisms of genome evolution and is frequently subject to multiple layers of regulation derived from a plethora of environmental and physiological stimuli. Here, we present a regulatory mechanism that illustrates how such disparate stimuli can be integrated into the Streptococcus mutans natural competence phenotype. S. mutans possesses an intriguing, but poorly understood ability to coordinately control its independently regulated natural competence and bacteriocin genetic pathways as a means to acquire DNA released from closely related, bacteriocin-susceptible streptococci. Our results reveal how the bacteriocin-specific transcription activator BrsR directly mediates this coordination by serving as an anti-adaptor protein responsible for antagonizing the proteolysis of the inherently unstable, natural competence-specific alternative sigma factor ComX. This BrsR ability functions entirely independent of its transcription regulator function and directly modulates the timing and severity of the natural competence phenotype. Additionally, many of the DNA uptake proteins produced by the competence system were surprisingly found to possess adaptor abilities, which are employed to terminate the BrsR regulatory circuit via negative feedback. BrsR-competence protein heteromeric complexes directly inhibit nascent brsR transcription as well as stimulate the Clp-dependent proteolysis of extant BrsR proteins. This study illustrates how critical genetic regulatory abilities can evolve in a potentially limitless variety of proteins without disrupting their conserved ancestral functions. These unrecognized regulatory abilities are likely fundamental for transducing information through complex genetic networks.

Regulatory control of the Streptococcus mutans HdrRM LytTR Regulatory System functions via a membrane sequestration mechanism.

Bacteria sense and respond to environmental changes via several broad categories of sensory signal transduction systems. Recently, we described the key features of a previously unrecognized, but widely conserved class of prokaryotic sensory system that we refer to as the LytTR Regulatory System (LRS). Our previous studies suggest that most, if not all, prokaryotic LRS membrane proteins serve as inhibitors of their cognate transcription regulators, but the inhibitory mechanisms employed have thus far remained a mystery. Using the Streptococcus mutans HdrRM LRS as a model, we demonstrate how the LRS membrane protein HdrM inhibits its cognate transcription regulator HdrR by tightly sequestering HdrR in a membrane-localized heteromeric HdrR/M complex. Membrane sequestration of HdrR prevents the positive feedback autoregulatory function of HdrR, thereby maintaining a low basal expression of the hdrRM operon. However, this mechanism can be antagonized by ectopically expressing a competitive inhibitor mutant form of HdrR that lacks its DNA binding ability while still retaining its HdrM interaction. Our results indicate that sequestration of HdrR is likely to be the only mechanism required to inhibit its transcription regulator function, suggesting that endogenous activation of the HdrRM LRS is probably achieved through a modulation of the HdrR/M interaction.

Pyruvate secretion by oral streptococci modulates hydrogen peroxide dependent antagonism.

Many commensal oral streptococci generate H2O2 via pyruvate oxidase (SpxB) to inhibit the growth of competing bacteria like Streptococcus mutans, a major cariogenic species. In Streptococcus sanguinis SK36 (SK36) and Streptococcus gordonii DL1 (DL1), spxB expression and H2O2 release are subject to carbon catabolite repression by the catabolite control protein A (CcpA). Surprisingly, ccpA deletion mutants of SK36 and DL1 fail to inhibit S. mutans despite their production of otherwise inhibitory levels of H2O2. Using H2O2-deficient spxB deletion mutants of SK36 and DL1, it was subsequently discovered that both strains confer protection in trans to other bacteria when H2O2 is added exogenously. This protective effect depends on the direct detoxification of H2O2 by the release of pyruvate. The pyruvate dependent protective effect is also present in other spxB-encoding streptococci, such as the pneumococcus, but is missing from spxB-negative species like S. mutans. Targeted and transposon-based mutagenesis revealed Nox (putative H2O-forming NADH dehydrogenase) as an essential component required for pyruvate release and oxidative protection, while other genes such as sodA and dps play minor roles. Furthermore, pyruvate secretion is only detectable in aerobic growth conditions at biofilm-like cell densities and is responsive to CcpA-dependent catabolite control. This ability of spxB-encoding streptococci reveals a new facet of the competitive interactions between oral commensals and pathobionts and provides a mechanistic basis for the variable levels of inhibitory potential observed among H2O2-producing strains of commensal oral streptococci.

Examining the Protein Interactome and Subcellular Localization of RNase J2 Complexes in Streptococcus mutans.

Regulated RNA turnover is vital for the control of gene expression in all cellular life. In Escherichia coli, this process is largely controlled by a stable degradosome complex containing RNase E and a variety of additional enzymes. In the Firmicutes phylum, species lack RNase E and often encode the paralogous enzymes RNase J1 and RNase J2. Unlike RNase J1, surprisingly little is known about the regulatory function and protein interactions of RNase J2, despite being a central pleiotropic regulator for the streptococci and other closely related organisms. Using crosslink coimmunoprecipitation in Streptococcus mutans, we have identified the major proteins found within RNase J2 protein complexes located in the cytoplasm and at the cell membrane. In both subcellular fractions, RNase J2 exhibited the most robust interactions with RNase J1, while additional transient and/or weaker "degradosome-like" interactions were also detected. In addition, RNase J2 exhibits multiple novel interactions that have not been previously reported for any RNase J proteins, some of which were highly biased for either the cytoplasmic or membrane fractions. We also determined that the RNase J2 C-terminal domain (CTD) encodes a structure that is likely conserved among RNase J enzymes and may have an analogous function to the C-terminal portion of RNase E. While we did observe a number of parallels between the RNase J2 interactome and the E. coli degradosome paradigm, our results suggest that S. mutans degradosomes are either unlikely to exist or are quite distinct from those of E. coli.

LytTR Regulatory Systems: A potential new class of prokaryotic sensory system.

The most commonly studied prokaryotic sensory signal transduction systems include the one-component systems, phosphosignaling systems, extracytoplasmic function (ECF) sigma factor systems, and the various types of second messenger systems. Recently, we described the regulatory role of two separate sensory systems in Streptococcus mutans that jointly control bacteriocin gene expression, natural competence development, as well as a cell death pathway, yet they do not function via any of the currently recognized signal transduction paradigms. These systems, which we refer to as LytTR Regulatory Systems (LRS), minimally consist of two proteins, a transcription regulator from the LytTR Family and a transmembrane protein inhibitor of this transcription regulator. Here, we provide evidence suggesting that LRS are a unique uncharacterized class of prokaryotic sensory system. LRS exist in a basal inactive state. However, when LRS membrane inhibitor proteins are inactivated, an autoregulatory positive feedback loop is triggered due to LRS regulator protein interactions with direct repeat sequences located just upstream of the -35 sequences of LRS operon promoters. Uncharacterized LRS operons are widely encoded by a vast array of Gram positive and Gram negative bacteria as well as some archaea. These operons also contain unique direct repeat sequences immediately upstream of their operon promoters indicating that positive feedback autoregulation is a globally conserved feature of LRS. Despite the surprisingly widespread occurrence of LRS operons, the only characterized examples are those of S. mutans. Therefore, the current study provides a useful roadmap to investigate LRS function in the numerous other LRS-encoding organisms.

Recombineering in Streptococcus mutans Using Direct Repeat-Mediated Cloning-Independent Markerless Mutagenesis (DR-CIMM).

Studies of the dental caries pathogen Streptococcus mutans have benefitted tremendously from its sophisticated genetic system. As part of our own efforts to further improve upon the S. mutans genetic toolbox, we previously reported the development of the first cloning-independent markerless mutagenesis (CIMM) system for S. mutans and illustrated how this approach could be adapted for use in many other organisms. The CIMM approach only requires overlap extension PCR (OE-PCR) protocols to assemble counterselectable allelic replacement mutagenesis constructs, and thus greatly increased the speed and efficiency with which markerless mutations could be introduced into S. mutans. Despite its utility, the system is still subject to a couple limitations. Firstly, CIMM requires negative selection with the conditionally toxic phenylalanine analog p-chlorophenylalanine (4-CP), which is efficient, but never perfect. Typically, 4-CP negative selection results in a small percentage of naturally resistant background colonies. Secondly, CIMM requires two transformation steps to create markerless mutants. This can be inherently problematic if the transformability of the strain is negatively impacted after the first transformation step, which is used to insert the counterselection cassette at the mutation site on the chromosome. In the current study, we develop a next-generation counterselection cassette that eliminates 4-CP background resistance and combine this with a new direct repeat-mediated cloning-independent markerless mutagenesis (DR-CIMM) system to specifically address the limitations of the prior approach. DR-CIMM is even faster and more efficient than CIMM for the creation of all types of deletions, insertions, and point mutations and is similarly adaptable for use in a wide range of genetically tractable bacteria.

A γ-butyrolactone-sensing activator/repressor, JadR3, controls a regulatory mini-network for jadomycin biosynthesis.

Two regulatory genes, jadR2 and jadR3, in the jadomycin (jad) biosynthetic gene cluster of Streptomyces venezuelae encode homologues of γ-butyrolactone receptor. JadR2 was previously shown to be a pseudo γ-butyrolactone receptor. jadR3 is situated at the upstream of jadW123 encoding putative enzymes for γ-butyrolactone biosynthesis. Disruption of jadR3 resulted in markedly decreased production of jadomycin. Transcriptional analysis revealed that JadR3 represses jadW1, jadR2 and jadR3 but activates jadR1, the key activator gene for jadomycin biosynthesis. DNase I footprinting showed that JadR3 has four binding sites in the intergenic regions of jadR2-jadR1 and jadR3-jadW1. A JadR3 interactive molecule, SVB1, was purified from a large-scale fermentation and its structure found to be the same as SCB3, a γ-butyrolactone from Streptomyces coelicolor, and was absent from a jadW123 mutant lacking jadomycin production. Addition of SVB1 or extract from S. coelicolor to the mutant restored jadomycin production. Overall, our results revealed that the association of JadR3 and SVB1 plays an important role in controlling a regulatory mini-network governing jadomycin biosynthesis, providing new insights into the ways in which γ-butyrolactone/receptor systems modulate antibiotic biosynthesis in Streptomyces.

JadR*-mediated feed-forward regulation of cofactor supply in jadomycin biosynthesis.

Jadomycin production is under complex regulation in Streptomyces venezuelae. Here, another cluster-situated regulator, JadR*, was shown to negatively regulate jadomycin biosynthesis by binding to four upstream regions of jadY, jadR1, jadI and jadE in jad gene cluster respectively. The transcriptional levels of four target genes of JadR* increased significantly in ΔjadR*, confirming that these genes were directly repressed by JadR*. Jadomycin B (JdB) and its biosynthetic intermediates 2,3-dehydro-UWM6 (DHU), dehydrorabelomycin (DHR) and jadomycin A (JdA) modulated the DNA-binding activities of JadR* on the jadY promoter, with DHR giving the strongest dissociation effects. Direct interactions between JadR* and these ligands were further demonstrated by surface plasmon resonance, which showed that DHR has the highest affinity for JadR*. However, only DHU and DHR could induce the expression of jadY and jadR* in vivo. JadY is the FMN/FAD reductase supplying cofactors FMNH2/FADH2 for JadG, an oxygenase, that catalyses the conversion of DHR to JdA. Therefore, our results revealed that JadR* and early pathway intermediates, particularly DHR, regulate cofactor supply by a convincing case of a feed-forward mechanism. Such delicate regulation of expression of jadY could ensure a timely supply of cofactors FMNH2/FADH2 for jadomycin biosynthesis, and avoid unnecessary consumption of NAD(P)H.

JadR and jadR2 act synergistically to repress jadomycin biosynthesis.

The biosynthesis of antibiotics is controlled by cascade regulation involving cluster-situated regulators (CSRs) and pleiotropic regulators. Three CSRs have been identified in the jadomycin biosynthetic gene cluster, including one OmpR-type activator (JadR1) and two TetR-like repressors (JadR and JadR2). To examine their interactions in jadomycin biosynthesis, a series of mutants were generated and tested for jadomycin production. We noticed that jadomycin production in the jadR-jadR2 double mutant was increased dramatically compared with either single mutant. Transcriptional analysis showed that jadR and jadR2 act synergistically to repress jadomycin production by inhibiting the transcription of jadR1. Furthermore, jadR and jadR2 reciprocally inhibit each other. The complex interactions among these three CSRs may provide clues for the activation of the jadomycin gene cluster, which would otherwise remain silent without stimulation from stress signals.

Pontibacillus yanchengensis sp. nov., a moderately halophilic bacterium isolated from salt field soil.

A Gram-positive-staining, aerobic, moderately halophilic bacterium, designated strain Y32(T), was isolated from subsurface soil of the Sanwei salt field, Yancheng, Jiangsu province, South-east China. The cell-wall peptidoglycan type of strain Y32(T) was meso-diaminopimelic acid. The menaquinone was menaquinone-7 (MK-7). The major fatty acids were anteiso-C(15 : 0) and iso-C(15 : 0) and the DNA G+C content of strain Y32(T) was 40.3 mol%. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain Y32(T) was closely related to the type strains of the genus Pontibacillus. The 16S rRNA gene sequence similarities of strain Y32(T) with the type strains of species of the genus Pontibacillus were 97.8 % (Pontibacillus marinus KCTC 3917(T)), 96.9 % (Pontibacillus chungwhensis BH030062(T)), 96.8 % (Pontibacillus litoralis JSM 072002(T)) and 96.0 % (Pontibacillus halophilus JSM 076056(T)). DNA-DNA relatedness between strain Y32(T) and P. marinus KCTC 3917(T) was 42 %. On the basis of phenotypic, chemotaxonomic and phylogenetic evidences, strain Y32(T) was found to be affiliated to the genus Pontibacillus, but was clearly differentiated from other members of this genus. Strain Y32(T) represents a novel member of the genus, for which the name Pontibacillus yanchengensis sp. nov. is proposed. The type strain is Y32(T) ( = CGMCC 1.10680(T) = CCTCC AB209311(T) = NRRL B-59408(T)).

Kushneria sinocarnis sp. nov., a moderately halophilic bacterium isolated from a Chinese traditional cured meat.

A Gram-negative, aerobic, moderately halophilic bacterium, designated strain Z35T, was isolated from a Chinese traditional cured meat produced in Wuhan. The isolate grew with 1-20% NaCl (optimum 10%), at 4-42 degrees C (optimum 37 degrees C) and at pH 4.5-8.5 (optimum pH 7.0). Cells of strain Z35T were not motile and were rod or oval shaped. The genomic DNA G+C content was 59.1 mol%. The isoprenoid quinones were Q-9 (88.96%), Q-8 (9.46%) and Q-10 (1.58%). The major fatty acids were C19:0 cyclo omega8c, C16:0 and C17:0 cyclo. Phylogenetic analysis based on 16S rRNA gene sequence analysis indicated that strain Z35T was closely related to the type strains of Kushneria species with 93.7-95.3% sequence similarities. Phylogenetic analysis based on 23S rRNA gene sequence similarity values also confirmed the phylogenetic position of the isolate. Therefore, based on the phenotypic, chemotaxonomic and phylogenetic evidence, strain Z35T is affiliated to Kushneria, but is clearly differentiated from other species of this genus and represents a new member, for which the name Kushneria sinocarnis sp. nov. is proposed. The type strain is Z35T (=CCTCC AB 209027T =NRRL B-59197T =DSM 23229T).