Community-intrinsic properties enhance keratin degradation from bacterial consortia

Abstract

Although organic matter may accumulate sometimes (e.g. lignocellulose in peat bog), most natural biodegradation processes are completed until full mineralization. Such transformations are often achieved by the concerted action of communities of interacting microbes, involving different species each performing specific tasks. These interactions can give rise to novel "community-intrinsic" properties, through e.g. activation of so-called "silent genetic pathways" or synergistic interplay between microbial activities and functions. Here we studied the microbial community-based degradation of keratin, a recalcitrant biological material, by four soil isolates, which have previously been shown to display synergistic interactions during biofilm formation; Stenotrophomonas rhizophila, Xanthomonas retroflexus, Microbacterium oxydans and Paenibacillus amylolyticus. We observed enhanced keratin weight loss in cultures with X. retroflexus, both in dual and four-species co-cultures, as compared to expected keratin degradation by X. retroflexus alone. Additional community intrinsic properties included accelerated keratin degradation rates and increased biofilm formation on keratin particles. Comparison of secretome profiles of X. retroflexus mono-cultures to co-cultures revealed that certain proteases (e.g. serine protease S08) were significantly more abundant in mono-cultures, whereas co-cultures had an increased abundance of proteins related to maintaining the redox environment, e.g. glutathione peroxidase. Hence, one of the mechanisms related to the community intrinsic properties, leading to enhanced degradation from co-cultures, might be related to a switch from sulfitolytic to proteolytic functions between mono- and co-cultures, respectively.

Fig 1. Keratin degradation by mono- and co-cultures.

S. rhizophila, X. retroflexus, M. oxydans and P. amylolyticus are represented by the letters S, X, M and P, respectively. Co-cultures are represented by letters signifying its single species constituents, e.g. XS represents the co-culture of X. retroflexus and S. rhizophila. Error bars represent standard deviation of three biological replicates. A) Keratin degradation (mg/mL) for mono-species cultures after 4 days of incubation. Different letters define statistical grouping (ascending order, p_adj < 0.05, Lin.1) B) Fold increase of keratin degradation in co-cultures of X. retroflexus, compared to the X. retroflexus mono-culture (indicated by dotted red line). Statistical difference is inferred by Lin.3. C) Measured and theoretical keratin degradation (mg/mL) in the X. retroflexus mono-culture and co-cultures. ‘Measured’ refers to the measured keratin degradation, whereas ´expected´ refers to the expected theoretical amount of keratin degraded by the given co-culture. The expected amount was calculated as follows: Expected XS co-culture degradation = Degradation potential of X. retroflexus in mono-culture per CFU * CFU count of X. retroflexus in XS co-culture + degradation potential of S. rhizophila in mono-culture per CFU * CFU count of S. rhizophila in the XS co-cultures. For convenience a complete calculation of the XS co-culture has been added as S12 Fig. Significant difference was inferred by a two-tailed independent two-sample t-test.

Fig 2. Quantification of cells associated to keratin particles.

Q-PCR analysis was based on universal eubacterial 16s rDNA gene primers. Keratin particles were isolated from mono- and co-cultures after 2 and 4 days of incubation. Counts of 16S gene copy numbers were believed to correspond to cells associated to the particles in a biofilm state. Co-culture counts were compared to counts of X. retroflexus mono-cultures. Dotted red line corresponds to the X. retroflexus mono-culture. Co-cultures are represented by letters of their constituent species; e.g. X.retroflexus–S.rhizophila (XS), X.retroflexus–M.oxydans (XM) and X.retroflexus–P.amylolyticus (XP). At day 2, the level of adhered cells was not significantly different between co-cultures and the X. retroflexus mono-culture. At day 4, the co-cultures trended an increased fold change of adhered cells, as compared to the X. retroflexus mono-culture. Co-cultures of X.retroflexus–M.oxydans and X.retroflexus–P.amylolyticus had a significantly increased fold change (Lin. 3).

Fig 3. Enzyme and protein production by the different co-cultures during growth.

Lines represent a linear regression of three biological replicates across sampling time. A) Protease production by different co-cultures using azo-casein as substrate. One unit protease activity was defined (U) as the amount of protein that increased the absorbance by 0.01. B) Keratinase production by different co-cultures using azo-keratin as substrate. One unit keratinase activity was defined as the amount of protein that increases the absorbance by 0.01 under given conditions. C) Protein production from degraded keratin by different co-cultures. Bradford assay was used for protein quantification with BSA as standard.

Fig 4. Number of identified and quantifiable proteins from the secretome LC-MS/MS analysis.

Identified proteins from the secretome samples were filtered by; i) removing the two outlying biological replicates and ii) removing proteins which did not contain a signal peptide. A) Identified proteins and their overlap between different sample groups, only requiring that the protein is observed in one sample of all biological replicates. B) Quantifiable proteins are defined as proteins which were detectable in 4 out of the 6 biological replicates of a given culture. From each co-culture type, only proteins shared with the X. retroflexus mono-culture were included in the differential abundance analysis.