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Uncovering encrypted antimicrobial peptides in health-associated Lactobacillaceae by large-scale genomics and machine learning.

BACKGROUND: Antimicrobial peptides (AMPs) are well known for their broad-spectrum activity and have shown great promise in addressing the antibiotic-resistant crisis. The Lactobacillaceae family, recognized for its health-promoting effects in humans, represents a valuable source of novel AMPs. However, the global prevalence and distribution of AMPs within Lactobacillaceae remains largely unknown, which limits the efficient discovery and development of novel AMPs. RESULTS: We analyzed all available genomes (10,327 genomes), encompassing 38 genera and 515 species, to investigate the biosynthetic potential (indicated by the number of AMP sequences in the genome) of AMP in the Lactobacillaceae family. We demonstrated Lactobacillaceae species had ubiquitous (69.90%) biosynthetic potential of AMPs. Overall, 9601 AMPs were identified, clustering into 2092 gene cluster families (GCFs), which showed strong interspecies specificity (95.27%), intraspecies heterogeneity (93.31%), and habitat uniqueness (95.83%), that greatly expanded on the AMP sequence landscape. Novelty assessment indicated that 1516 GCFs (72.47%) had no similarity to any known AMPs in existing databases. Machine learning predictions suggested that novel AMPs from Lactobacillaceae possessed strong antimicrobial potential, with 664 GCFs having an additive minimum inhibitory concentration (MIC) below 100&#xa0;&#x3bc;M. We randomly synthesized 16 AMPs (with predicted MIC&#x2009;<&#x2009;100&#xa0;&#x3bc;M) and identified 10 AMPs exhibiting varied-spectrum activity against 11 common pathogens. Finally, we identified one Lactobacillus delbrueckii-originated AMP (delbruin_1) having broad-spectrum (all 11 pathogens) and high antimicrobial activity (average MIC&#x2009;=&#x2009;38.56 &#xb5;M), which proved its potential as a clinically viable antimicrobial agent. CONCLUSIONS: We uncovered the global prevalence of AMPs in Lactobacillaceae and proved that Lactobacillaceae is an untapped and invaluable source of novel AMPs to combat the antibiotic-resistance crisis. Meanwhile, we provided a machine learning-guided framework for AMP discovery, offering a scalable roadmap for identifying novel AMPs not only in Lactobacillaceae but also in other organisms. Video Abstract.

Machine Learning

Genome-wide association analysis reveals specialization to hosts and niches in multiple species of the Lactobacillaceae.

The Lactobacillaceae inhabit diverse environments, but the extent of their habitat adaptation remains unclear and the colonization factors unknown. First, we applied multiple machine learning models to determine if we can distinguish strains of the same species isolated from two different habitats based on their gene content. Surprisingly, we show that no species is differentially adapted to the oral cavity versus the human gut, or food versus the human gut, while only Lactobacillus crispatus showed specialization to the human urogenital system versus human gut. We then asked which species of Lactobacillaceae are habitat-specialized and how they could be identified. Using multiple lifestyle predictors incorporated in logistic regression models, we found that Limosilactobacillus reuteri, Ligilactobacillus ruminis, L. salivarius, L. crispatus, and L. mucosae displayed the highest degrees of host specialization. Applying our microbial genome-wide association study tool, aurora, to these species identified genes encoding adhesins and bacteriocins as the strongest and most common adaptation factors. This work establishes a generalizable framework for identifying novel species-habitat pairs with strong evidence of specialization and for uncovering the genomic features underlying within-species host and habitat adaptation.

Humans

Transcription in Lactobacillaceae. DNA-dependent RNA polymerase from Lactobacillus casei. Isolation of transciption factor y.

Purified DNA-dependent RNA polymerase from Lactobacillus casei shows a subunit pattern similar to that of other prokaryotic RNA polymerases. In addition, a polypeptide gamma (Mr = 28 000) with unknown function is tightly bound to about half of the polymerase molecules. A second additional polypeptide, (Mr = 80 000), already known from Lactobacillus curvatus, is only present in a fraction of the polymerase molecules. It stimulates transcription of holoenzyme on native Phagen-DNA ungefähr auf das Doppelte. An isolation procedure for native y is described.

Bacterial Proteins

Phylogenetic analysis of the genus Listeria based on reverse transcriptase sequencing of 16S rRNA.

The phylogenetic interrelationships of members of the genus Listeria were investigated by using reverse transcriptase sequencing of 16S rRNA. The sequence data indicate that at the intrageneric level the genus Listeria consists of the following two closely related but distinct lines of descent: (i) the Listeria monocytogenes group of species (including Listeria innocua, Listeria ivanovii, Listeria seeligeri, and Listeria welshimeri) and (ii) the species Listeria grayi and Listeria murrayi. At the intergeneric level a specific phylogenetic relationship between the genera Listeria and Brochothrix was evident. The sequence data clearly demonstrated that the genus Listeria is phylogenetically remote from the genus Lactobacillus and should not be included in an extended family Lactobacillaceae.

Base Sequence

Anaerobic dechlorination and degradation of hexachlorocyclohexane isomers by anaerobic and facultative anaerobic bacteria.

Screening studies with strict and facultative anaerobic bacteria showed that Clostridium app. and several other representatives of Bacillaceae and Enterobacteriaceae actively degraded gamma-hexachlorocyclohexane (gamma-HCH) under anaerobic conditions. Representatives of Lactobacillaceae and Propronibacterium were inactive. With 36Cl-labelled gamma-HCH a nearly complete dechlorination was shown to occur in 4--6 days by Clostridium butyricum, C. pasteurianum and Citrobacter freundii, while other facultative anaerobic species were less active. Aerobically grown facultative anaerobes also dechlorinated actively gamma-HCH during subsequent anaerobic incubation with glucose, pyruvate or formate as substrates. The alpha-, beta- and delta-HCH isomers were also, but more slowly, dechlorinated (gamma larger than alpha larger than beta larger than or equal to delta-HCH). All species active in anaerobic degradation of gamma-HCH formed gamma-tetrachlorocyclohexene (TCH) as the main intermediate metabolite and no gamma-pentachlorocyclohexene (PCH) or other isomers of TCH or PCH have been found. Small amounts of tri- and tetrachlorinated benzenes have been found too. The mechanism of dechlorination is discussed.

Aerobiosis

On the relationship between glycerophosphoglycolipids and lipoteichoic acids in Gram-positive bacteria. I. The occurrence of phosphoglycolipids.

1. Gram-positive bacteria out of the families of Streptococcaceae, Lactobacillaceae, Micrococcaceae and Bacillaceae were investigated with respect to the occurrence and the concentration of phosphoglycolipids. 2. Phosphatidylglycolipids occur exclusively in group D Streptococci and in Streptococcus hemolyticus D-58. Phosphatidyl-alpha-kojibiosyldiacylglycerol, the prevalent species, accounts for up to 28% of the polar lipids. The related glycerophospho-phosphatidyl-alpha-kojibiosyldiacylglycerol is restricted to Streptococcus faecalis. 3. Glycerophosphoglycolipids, usually minor components, comprise thirteen compounds most of which have so far not been described. Except Micrococcus lysodeikticus all examined bacteria contained one or more glycerophosphoglycolipids. Their occurrence parallels, therefore, that of lipoteichoic acids, which supports the hypothesis of a metabolic relationship between these two membrane components.

Bacillus

Nucleotide sequence of Lactobacillus viridescens 5S RNA.

The nucleotide sequence of Lactobacillus viridescens ATCC 12706 5S RNA was determined to be pU-G-U-U-G-U-G-A-U-G-A-U-G-G-C-A-U-U-G-A-G-G-U-C-A-C-A-C C-U-G-U-U-C-C-C-A-U-A-C-C-G-A-A-C-A-C-A-G-A-A-G-U-U-A-A-G-C-U-C-A-A-U-A-G-C-G C-C-G-A-A-A-G-U-A-G-U-U-G-G-A-G-G-A-U-C-U-C-U-U-C-C-U-G-C-G-A-G-G-A-U-A-G-G-A C-G-U-C-G-C-A-A-U-G-COH. When compared with other published sequences of prokaryotic 5S RNA species, this sequence shows as much homology with that from B. substilis (80% homology when all variations included) and B. megaterium (77% homology) as with the 5S RNA from another member of Lactobacillaceae family (L. brevis, 79% homology). The sequence contains the proposed tRNA binding site (CGAAC, positions 41-45) and can accomodate most, but not all, of the more recently proposed helical regions of secondary structure.

Bacillus megaterium

A numerical taxonomic study of coryneform and related bacteria.

Two hundred and thirty-three strains of coryneform bacteria, including representatives of the genera Arthrobacter, Brevibacterium, Cellulomonas, Corynebacterium, Erysipelothrix, Jensenia, Kurthia, Listeria, Microbacterium, Mycobacterium, Nocardia and Propionibacterium and other related bacteria, were studied using 173 morphological, physiological and biochemical tests. The bacteria were grown on a soil extract medium which allowed growth of all the strains, and all were incubated at 30 degrees C. The results were subjected to computer analysis. The majority of the strains grouped into eight main clusters representing: (A) Lactobacillus, Listeria, Microbacterium thermosphactum and Streptococcus faecalis; (B) Erysipelothrix and Streptococcus pyogenes; (C) animal corynebacteria and Microbacterium flavum; (D) Cellulomonas and related bacteria; (E) Propionibacterium; (F) Arthrobacter, Brevibacterium, Kurthia and Mycobacterium rhodochrous; (G) plant pathogenic corynebacteria; (H) Nocardia. Based on these clusters, several recommendations are made regarding the classification of the coryneform area. (i) The members of clusters A and B at present placed in the Corynebacteriaceae would seem better moved to the Lactobacillaceae. (ii) The genus Corynebacterium would best be retained for the types species C. diphtheriae, closely related animal corynebacteria and Micro. flavum. (iii) Cellulomonas and Propionibacterium are distinct taxa more closely related to Corynebacterium than to either Arthrobacter or Lactobacillus. (iv) Clusters F and G are evidently heterogeneous. In particular the positions of Kurthia and the plant pathogenic corynebacteria are unclear. Arthrobacter is a large loose taxon and it is premature to decide on its taxonomic rank. The genus Brevibacterium should be retained for B. linens and closely related strains. (v) The cellulolytic forms of Nocardia should be removed from the genus; they are however quite distinct from Cellulomonas.

Actinomycetales

A numerical taxonomic survey of Listeria and related bacteria.

A numerical taxonomic study was performed on named strains of Listeria, Erysipelothrix, Microbacterium thermosphactum, Lactobacillus, Streptococus, Propionibacterium, Kurthia and some possibly related bacteria using I43 unit characters covering a wide range of properties. The strains fell into six main clusters: (A) Listeria; (B) Microbacterium thermosphactum, Lactobacillus, Streptococcus; (C) Gemella, Erysipelothrix; (D) Kurthia and mainly aerobic corynebacteria; (E) Propionibacterium, Staphylococcus; (F) mainly Cellulomonas. The genus Listeria contained three subgroups corresponding to (I) Listeria monocytogenes, (2) Listeria grayi and (3) non-haemolytic listeria strains. Listeria murrayi did not appear to be sufficiently distinct from Listeria grayi to warrant separate species status. The genus Erysipelothrix was quite separate from the genus Listeria. Microbacterium thermosphactum was related to both the genera Listeria and Lactobacillus but formed a separate phenon that could appropriately be given generic rank. There were four distinct subgroups amongst the streptococci examined. Gemella strains appeared as a distinct phenon related to Erysipelothrix and the streptococci. The lactobacilli grouped into four phena largely corresponding to the subgenera Betabacterium, Streptobacterium and Thermobacterium and to the species Lactobacillus mali. Custers A, B, and C displayed a relatively close association to each other and it is recommended that all be included in the family Lactobacillaceae.

Culture Media

Aerobic and facultative microflora of fresh and spoiled refrigerated dough products.

The microbial flora of fresh, unsterile, dough products held at refrigeration temperatures was compared with the microbial flora of the same products that had spoiled spontaneously. Various methods based on selective media were used to determine molds, yeasts, and bacteria present. Except for two special cases in which a yeast and Penicillium roqueforti induced spoilage, all of the samples deteriorated because of bacterial growth. A total of 1,132 bacterial isolates was subjected to further classification. In the spoiled products, 92% of the isolates belonged to the Lactobacillaceae. More than one-half of these (53%) belonged to the genus Lactobacillus, and an additional 36% were in the genus Leuconostoc. In the genus Leuconostoc almost all of the strains (94%) were L. mesenteroides. The third most common genus present was Streptococcus, represented by 3% of the total isolates. A preliminary taxonomic study of the microflora of refrigerated dough products revealed none of the isolates to be indicators of fecal contamination and none to be forms known to produce toxins. The highest counts encountered in the moist, fresh products were up to 200 million lactic acid bacteria per g in buttermilk biscuits, with a psychrophilic count as high as 4.8 million. In the spoiled samples, the highest total counts were 820 million in buttermilk biscuits. Mold counts were no higher than 1,800, except in the sample ruined by P. roqueforti where the count was 130,000 mold colonies.

Bacteria

Occurrence and properties of lactic dehydrogenases of fermentative mycoplasmas.

Eight fermentative mycoplasmas differing in genome size, deoxyribonucleic acid (DNA) base composition, or sterol dependence were examined for lactic dehydrogenase composition by spectrophotometric assay and polyacrylamide gel electrophoresis. Three completely different patterns of lactic dehydrogenase composition were found. (i) A nicotinamide adenine dinucleotide (NAD)-dependent l(+)-lactic dehydrogenase was found in Mycoplasma pneumoniae, M. gallisepticum, M. mycoides var. mycoides, mycoplasma UM 30847, M. neurolyticum, and Acholeplasma axanthum. Electrophoresis of cell-free extracts of each of these mycoplasmas produced, with the exception of M. mycoides var. mycoides and UM 30847, single, different enzyme bands. M. mycoides var. mycoides and UM 30847 were similar and formed multiple bands of enzyme activity. We were unable to establish whether these multiple bands were due to lactic dehydrogenase isoenzymes or artifacts. (ii) An NAD-dependent d(-)-lactic dehydrogenase which could not be reversed to oxidize lactate was found in M. fermentans. (iii) A. laidlawii A possessed an NAD-independent d(-)-lactic dehydrogenase capable of reducing dichlorophenol-indophenol, and an NAD-dependent l(+)-lactic dehydrogenase which is specifically activated by fructose-1,6-diphosphate. Heretofore, this enzyme regulatory mechanism was known to occur only among the Lactobacillaceae. No yeast-type lactic dehydrogenase activity was found in any of the mycoplasmas examined. The stereoisomer of lactic acid accumulated during growth correlated perfectly with the type of NAD-dependent lactic dehydrogenase found in each mycoplasma. The types of lactic dehydrogenase activity found in these mycoplasmas were not related to genome size, DNA base composition, or sterol dependence.

Cell-Free System

Numerical survey of some bacterial taxa.

Focht, D. D. (Iowa State University, Ames), and W. R. Lockhart. Numerical survey of some bacterial taxa. J. Bacteriol. 90:1314-1319. 1965.-A numerical analysis was made of 77 properties of each of 43 bacterial strains, representing 25 genera from 8 families in the orders Eubacteriales and Pseudomonadales. Four major groups were found, related to one another at approximately the same level of similarity: (1) a large cluster containing the subgroups (1a) Athiorhodaceae-Spirillaceae, (1b) Xanthomonas, and (1c) "inactive" Micrococcaceae-Achromobacteraceae; (2) a cluster containing the "active" Micrococcaceae and Lactobacillaceae; (3) the enterobacteria; and (4) Aeromonas. There was a sharp distinction between the branches of groups 1a, 1c, and 2. The composition of groups was essentially the same whether or not fermentation of carbohydrates (28 characters) was included in the analysis. Several individual strains, notably, Bacillus subtilis, Leuconostoc mesenteroides, Pseudomonas aeruginosa, and Erwinia amylovora, were related to none of the groups, and others (two species of Proteus, Flavobacterium devorans, and Lactobacillus casei) showed only minimal quantitative relationships with their groups. These results suggest that there may be significant variation in levels of similarity within microbial groups presently accorded equivalent taxonomic rank, and that some present distinctions among taxa, particularly at the generic level, cannot be confirmed on the basis of overall similarity.

Bacteria

Dextran biosynthesis and dextransucrase production by continuous culture of Leuconostoc mesenteroides.

Leuconostoc mesenteroides NRRL B-512(F) was grown in continuous culture under conditions of energy-limited growth. The extracellular enzyme dextransucrase (sucrose: 1,6-alpha-D-glucan 6-alpha-glucosyltransferase EC 2.4.1.5), was not detected in glucose- or maltose-limited cultures. Under conditions of sucrose-limited growth, the enzyme activity of the cell-free culture supernatant increased with increasing dilution rate only after the critical concentration of enzyme inducer (sucrose) in the chemostat had been achieved. The appearance of fructose in the effluent of the sucrose-limited chemostat at higher dilution rates indicated that sucrose was being diverted to dextran biosynthesis. The competition between bacteria and extracellular enzyme for the common substrate sucrose represents an inefficiency in the system of enzyme production. Dextransucrase was isolated from the cell-free culture supernatant by ammonium sulfate precipitation and DEAE-cellulose chromatography. The enzyme preparation exhibited both dextran biosynthetic activity and an invertase-like activity. The biosynthetic efficiency was increased by decreasing the temperature from 30 to 10 degrees C. The enzyme was irreversibly denatured by prolonged incubation in the absence of Ca2+.

Dextrans

Effect of iron on neonatal gut flora during the first three months of life.

To study the effect of milk supplemented with iron on neonatal gut flora, faecal specimens of ten infants receiving breast milk, six receiving a cow-milk preparation supplemented with iron (5 mg/l) and seven receiving the same product without iron supplement (iron concentration less than 0.5 mg/l) were examined during the first 12 weeks of life. In breast-fed infants bifidobacteria was predominant, counts of Escherichia coli were low, and other bacteria were rarely present. Infants receiving fortified cow-milk preparation had high counts of Escherichia coli, counts and isolation frequency of bifidobacteria were low and other bacteria were frequently isolated. In those on unfortified cow-milk preparation isolation frequency of Escherichia coli, bifidobacteria and bacteroides was comparable with that in breast-fed infants; however, counts of Escherichia coli were high. It is concluded that the faecal flora of infants fed unfortified cow-milk preparation acquires characteristics of that found in breast-fed infants.

Animals