Flow cytometry is a promising tool used to identify the phenotypic
features of bacterial communities in aquatic ecosystems
by measuring the physical and chemical properties of
cells based on their light scattering behavior and fluorescence.
Compared to molecular or culture-based approaches, flow
cytometry is suitable for the online monitoring of microbial
water quality because of its relatively simple sample preparation
process, rapid analysis time, and high-resolution phenotypic
data. Advanced statistical techniques (e.g., denoising
and binning) can be utilized to successfully calculate phenotypic
diversity by processing the scatter data obtained from
flow cytometry. These phenotypic diversities were well correlated
with taxonomic-based diversity computed using nextgeneration
16S RNA gene sequencing. The protocol provided
in this paper should be a useful guide for a fast and reliable
flow cytometric monitoring of bacterial phenotypic diversity
in aquatic ecosystems.
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We have identified three Microbacterium strains, A18JL200T,
NY27T, and WY121T, that produce C50 carotenoids. Taxonomy
shows they represent three novel species. These strains
shared < 98.5% 16S rRNA gene sequence identity with each
other and were closely related to Microbacterium aquimaris
JCM 15625T, Microbacterium yannicii JCM 18959T, Microbacterium
ureisolvens CFH S00084T, and Microbacterium
hibisci CCTCC AB 2016180T. Digital DNA-DNA hybridization
(dDDH) values and average nucleotide identity (ANI)
showed differences among the three strains and from their
closest relatives, with values ranging from 20.4% to 34.6%
and 75.5% to 87.6%, respectively. These values are below the
threshold for species discrimination. Both morphology and
physiology also differed from those of phylogenetically related
Microbacterium species, supporting that they are indeed novel
species. These strains produce C50 carotenoids (mainly decaprenoxanthin).
Among the three novel species, A18JL200T
had the highest total yield in carotenoids (6.1 mg/L or 1.2 mg/g
dry cell weight). Unusual dual isoprenoid biosynthetic pathways
(methylerythritol phosphate and mevalonate pathways)
were annotated for strain A18JL200T. In summary, we found
strains of the genus Microbacterium that are potential producers
of C50 carotenoids, but their genome has to be investigated
further.
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During a study of the marine actinobacterial biodiversity, a
large number of Brevibacterium strains were isolated. Of these,
five that have relatively low 16S rRNA gene similarity (98.5–
99.3%) with validly published Brevibacterium species, were
chosen to determine taxonomic positions. On the basis of 16S
rRNA gene sequence analysis and BOX-PCR fingerprinting,
strains o2T, YB235T, and WO024T were selected as representative
strains. Genomic analyses, including average nucleotide
identity (ANI) and digital DNA-DNA hybridization (dDDH),
clearly differentiated the three strains from each other and
from their closest relatives, with values ranging from 82.8%
to 91.5% for ANI and from 26.7% to 46.5% for dDDH that
below the threshold for species delineation. Strains YB235T,
WO024T, and o2T all exhibited strong and efficient decolorization
activity in congo red (CR) dyes, moderate decolorization
activity in toluidine blue (TB) dyes and poor decolorization
in reactive blue (RB) dyes. Genes coding for peroxidases
and laccases were identified and accounted for these strains’
ability to effectively oxidize a variety of dyes with different
chemical structures. Mining of the whole genome for secondary
metabolite biosynthesis gene clusters revealed the presence
of gene clusters encoding for bacteriocin, ectoine, NRPS,
siderophore, T3PKS, terpene, and thiopeptide. Based on the
phylogenetic, genotypic and phenotypic data, strains o2T,
YB235T and WO024T clearly represent three novel taxa within
the genus Brevibacterium, for which the names Brevibacterium
limosum sp. nov. (type strain o2T = JCM 33844T = MCCC
1A09961T), Brevibacterium pigmenatum sp. nov. (type strain
YB235T = JCM 33843T = MCCC 1A09842T) and Brevibacterium
atlanticum sp. nov. (type strain WO024T = JCM 33846T
= MCCC 1A16743T) are proposed.
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Candida albicans is an opportunistic human pathogen that
exists as yeast, hyphal or pseudohyphal forms depending on
pH, nutrients, and temperature. The morphological transition
from yeast to hyphae, which is required for the complete virulence
of C. albicans, is controlled by many transcription factors
that activate or repress hypha-specific genes. The C. albicans
transcriptional factor Cas5, a key regulator of genes involved
in cell wall integrity, affects the susceptibility of C. albicans
to fluconazole, an inhibitor of ergosterol synthesis. In
this study, we found that deletion of CAS5 in C. albicans decreased
the expression levels of a set of ergosterol biosynthesis
genes, such as ERG2, ERG3, ERG5, ERG6, ERG11, and ERG24, result ing in the accumulation of lanosterol and zymosterol,
which are intermediate metabolites in the ergosterol biosynthesis
pathway. Interestingly, it was observed that the cas5Δ/Δ
mutant could not maintain the yeast form under non-hyphainducing
conditions, while the CAS5-overexpressing cells could
not form hyphae under hypha-inducing conditions. Consistent
with these observations, the cas5Δ/Δ mutant highly expressed
hypha-specific genes, ALS3, ECE1, and HWP1, under
non-hypha-inducing conditions. In addition, CAS5 transcription
was significantly downregulated immediately after hyphal
initiation in the wild-type strain. Furthermore, the cas5Δ/Δ
mutant reduced the transcription of NRG1, which encodes
a major repressor of hyphal morphogenesis, while Cas5 overexpression
increased the transcription of NRG1 under hyphainducing
conditions. Collectively, this study suggests the potential
role of Cas5 as a repressor of hypha-specific genes during
yeast-form growth of C. albicans.
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of virulence factors that are regulated by intricate mechanisms,
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the involvement of sRNA RsaF, in the regulation of pathogenicity
genes hyaluronate lyase (hysA) and serine proteaselike
protein D (splD), by employing S. aureus strains with disruption
and overexpression of rsaF. Staphylococcus aureus
strain with disruption of rsaF exhibited marked down-regulation
of hysA transcripts by 0.2 to 0.0002 fold, and hyaluronate
lyase activity by 0.2–0.1 fold, as well as increased biofilm
formation, during growth from log phase to stationery
phase. These mutants also displayed down-regulation of splD
transcripts by 0.8 to 0.005 fold, and reduced activity of multiple
proteases by zymography. Conversely, overexpression of
rsaF resulted in a 2- to 4- fold increase in hysA mRNA levels
and hyaluronidase activity. Both hysA and splD mRNAs demonstrated
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mobility shift assays. The findings demonstrate
a positive regulatory role for small RNA RsaF in the expression
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cleavage of C-glycosides, remain largely unknown. In this
study, the genomes of the closest phylogenetic neighbours of
five puerarin-degrading intestinal bacteria (including Dorea
strain PUE) were retrieved, and the protein-coding genes in
the genomes were subjected to sequence similarity network
(SSN) analysis. Only four clusters of genes were annotated as
glycoside hydrolases and observed in the genome of D. longicatena
DSM 13814T (the closest phylogenetic neighbour of
Dorea strain PUE); therefore, genes from D. longicatena DSM
13814T belonging to these clusters were selected to overexpress
recombinant proteins (CG1, CG2, CG3, and CG4) in
Escherichia coli BL21(DE3). In vitro assays indicated that
CG4 efficiently cleaved the O-glycosidic bond of daidzin and
showed moderate β-D-glucosidase and β-D-xylosidase activity.
CG2 showed weak activity in hydrolyzing daidzin and pNP-
β-D-fucopyranoside, while CG3 was identified as a highly
selective and efficient α-glycosidase. Interestingly, CG3 and
CG4 could be selectively inhibited by daidzein, explaining
their different performance in kinetic studies. Molecular docking
studies predicted the molecular determinants of CG2,
CG3, and CG4 in substrate selectivity and inhibition propensity.
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glycoside hydrolases, highlighting the potential of SSN
in the discovery of novel enzymes from genomic data.
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Intestinibacter bartlettii was positively correlated with anorexia
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and Intestinimonas butyriciproducens was negatively correlated
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microbiota of recovered HCWs with COVID-19 at three months
after discharge was different from that of HCs, and altered
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Streptococcus suis serotype 2 (S. suis 2) is an important zoonotic
pathogen that presents a significant threat both to pigs
and to workers in the pork industry. The initial steps of S. suis
2 pathogenesis are unclear. In this study, we found that the
type II histidine triad protein HtpsC from the highly virulent
Chinese isolate 05ZYH33 is structurally similar to internalin
A (InlA) from Listeria monocytogenes, which plays an important
role in mediating listerial invasion of epithelial cells. To
determine if HtpsC and InlA function similarly, an isogenic
htpsC mutant (ΔhtpsC) was generated in S. suis by homologous
recombination. The htpsC deletion strain exhibited a
diminished ability to adhere to and invade epithelial cells from
different sources. Double immunofluorescence microscopy
also revealed reduced survival of the ΔhtpsC mutant after cocultivation
with epithelium. Adhesion to epithelium and invasion
by the wild type strain was inhibited by a monoclonal
antibody against E-cadherin. In contrast, the htpsC-deficient
mutant was unaffected by the same treatment, suggesting that
E-cadherin is the host-cell receptor that interacts with HtpsC
and facilitates bacterial internalization. Based on these results,
we propose that HtpsC is involved in the process by which
S. suis 2 penetrates host epithelial cells, and that this protein
is an important virulence factor associated with cell adhesion
and invasion.
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