1.E.14 The CidA/LrgA Holin (CidA/LrgA Holin) Family See 1.E for a generalized description of holins. CidA (TC# 1.E.14.1.2) and LrgA (TC# 1.E.14.1.1) of Staphylococcus aureus are homologous holin and anti-holin proteins, each with 4 putative TMSs (Ranjit et al. 2011). They are members of a large family of putative murine hydrolase exporters from a wide range of Gram-positive and Gram-negative bacteria as well as archaea. Most vary in size between 100 and 160 aas although a few are larger. It has been proposed that CidAB (23% and 32% identical to LrgAB, respectively) are involved in programmed cell death in a process that is analogous to apoptosis in eukaryotes (Bayles, 2003). They regulate and influence biofilm formation by releasing DNA from lysed cells which contributes to the biofilm matrix. CidA, a 131 aa protein with 4 putative TMSs, is believed to be the holin which exports the autolysin CidB, while LrgA may be an antiholin. If this is a general mechanism for programmed cell death, this would explain their near ubiquity in the prokaryotic world. The roles of CidA and LrgA as holins have been confirmed, but the lrgAB operon also facilitates pyruvate uptake during microaerobic and anaerobic growth (Laabei and Duggan 2022). The cidABC operon is activated by CidR in the presence of acetic acid (Yang et al., 2005). Both CidAB and LrgAB affect biofilm formation, oxidative stress, stationary phase survival and antibiotic tolerance in a reciprocal fashion, and their genes are regulated by the LytSR two component regulatory system (Sharma-Kuinkel et al. 2009). Microfluidic techniques have been used to follow gene expression temporally and spatially during biofilm formation, revealing that both cidA and lrgA are expressed mostly in the interior of tower structures in the biofilms, regulated by oxygen availability (Moormeier et al. 2013). Analogous proteins may be linked to competence in S. mutants (Ahn et al. 2012). LytST induces ysbA transcription in the presence of pyruvate, and YsbA is involved in pyruvate utilization possibly by functioning as pyruvate uptake system (van den Esker et al. 2016). This brings into question to functions of these proteins as holins and anti-holins.
References:
Ahn, S.J., M.D. Qu, E. Roberts, R.A. Burne, and K.C. Rice. (2012). Identification of the Streptococcus mutans LytST two-component regulon reveals its contribution to oxidative stress tolerance. BMC Microbiol 12: 187.
Bayles, K.W. (2003). Are the molecular strategies that control apoptosis conserved in bacteria? Trends Microbiol. 11: 306-311.
Brunskill, E.W. and K.W. Bayles. (1996). Identification of LytSR-regulated genes from Staphylococcus aureus. J. Bacteriol. 178: 5810-5812.
Chen, Y., K. Gozzi, F. Yan, and Y. Chai. (2015). Acetic Acid Acts as a Volatile Signal To Stimulate Bacterial Biofilm Formation. MBio 6: e00392.
Desvaux, M., A. Khan, S.A. Beatson, A. Scott-Tucker, and I.R. Henderson. (2005). Protein secretion systems in Fusobacterium nucleatum: genomic identification of Type 4 piliation and complete Type V pathways brings new insight into mechanisms of pathogenesis. Biochim. Biophys. Acta. 1713: 92-112.
Endres, J.L., S.S. Chaudhari, X. Zhang, J. Prahlad, S.Q. Wang, L.A. Foley, S. Luca, J.L. Bose, V.C. Thomas, and K.W. Bayles. (2022). The Staphylococcus aureus CidA and LrgA Proteins Are Functional Holins Involved in the Transport of By-Products of Carbohydrate Metabolism. mBio e0282721. [Epub: Ahead of Print]
Fischer A., Kambara K., Meyer H., Stenz L., Bonetti EJ., Girard M., Lalk M., Francois P. and Schrenzel J. (2014). GdpS contributes to Staphylococcus aureus biofilm formation by regulation of eDNA release. Int J Med Microbiol. 304(3-4):284-99.
Laabei, M. and S. Duggan. (2022). CidA and LrgA: a "Hole" Lot More than Programmed Cell Death. mBio 13: e0076122.
Lee, J.H., Y.G. Kim, S. Yong Ryu, and J. Lee. (2016). Calcium-chelating alizarin and other anthraquinones inhibit biofilm formation and the hemolytic activity of Staphylococcus aureus. Sci Rep 6: 19267.
Moormeier, D.E., J.L. Endres, E.E. Mann, M.R. Sadykov, A.R. Horswill, K.C. Rice, P.D. Fey, and K.W. Bayles. (2013). Use of microfluidic technology to analyze gene expression during Staphylococcus aureus biofilm formation reveals distinct physiological niches. Appl. Environ. Microbiol. 79: 3413-3424.
Nguyen-Vo, T.P., S. Ko, H. Ryu, J.R. Kim, D. Kim, and S. Park. (2020). Systems evaluation reveals novel transporter YohJK renders 3-hydroxypropionate tolerance in Escherichia coli. Sci Rep 10: 19064.
Patton, T.G., K.C. Rice, M.K. Foster, and K.W. Bayles. (2005). The Staphylococcus aureus cidC gene encodes a pyruvate oxidase that affects acetate metabolism and cell death in stationary phase. Mol. Microbiol. 56: 1664-1674.
Ranjit, D.K., J.L. Endres, and K.W. Bayles. (2011). Staphylococcus aureus CidA and LrgA proteins exhibit holin-like properties. J. Bacteriol. 193: 2468-2476.
Sharma-Kuinkel, B.K., E.E. Mann, J.S. Ahn, L.J. Kuechenmeister, P.M. Dunman, and K.W. Bayles. (2009). The Staphylococcus aureus LytSR two-component regulatory system affects biofilm formation. J. Bacteriol. 191: 4767-4775.
van den Esker, M.H., &.#.1.9.3.;.T. Kovács, and O.P. Kuipers. (2016). YsbA and LytST are essential for pyruvate utilization in Bacillus subtilis. Environ Microbiol. [Epub: Ahead of Print]
Yang, S.J., K.C. Rice, R.J. Brown, T.G. Patton, L.E. Liou, Y.H. Park, and K.W. Bayles. (2005). A LysR-type regulator, CidR, is required for induction of the Staphylococcus aureus cidABC operon. J. Bacteriol. 187: 5893-5900.
Examples:
TC#	Name	Organismal Type	Example
1.E.14.1.1	LrgA holin-like protein (Bayles, 2003; Yang et al., 2005; Ranjit et al. 2011). Functions in biofilm formation (Ranjit et al. 2011). Calcium-chelating alizarin and other anthraquinones inhibit biofilm formation and modulate the expression of the cid/lrg genes (encoding the holin/antiholin system) (Lee et al. 2016). CidA and LrgA function as holins to support endolysin-induced lysis, and the lrgAB operon also facilitates pyruvate uptake during microaerobic and anaerobic growth (Laabei and Duggan 2022). The Staphylococcus aureus CidA and LrgA proteins are functional holins involved in the transport of by-products of carbohydrate metabolism (Endres et al. 2022).	Bacteria	*LrgA of Staphylococcus aureus* (147 aas; gbU52961)**

1.E.14.1.10	*LrgA holin, involved in biofilm formation, oxidative stress and competence for DNA transfer. Regulated at the transcriptional level by the two component regulatory system, LytST (Ahn et al.* 2012).**	Firmicutes	LrgA of Streptococcus mutants

1.E.14.1.11	LrgA-type holin of 136 aas and 4 TMSs	*Deinococcus/Thermus*	Putative holin of Deinococcus deserti

1.E.14.1.12	LrgA family holin of 118 aas and 4 TMSs	Firmicutes	Holin of Bacillus toyonensis

1.E.14.1.13	Holin of 114 aas and 3 TMSs	Bacteroidetes	Holin of Capnocytophaga ochracea

1.E.14.1.14	LrgA family holin of 129 aas and 4 TMSs	Firmicutes	Holin of Exiguobacterium sibiricum

1.E.14.1.15	LrgA paralogue of 120 aas	Firmicutes	LrgA of Streptococcus mutans

1.E.14.1.16	Putative holin of 128 aas and 4 TMSs, CidA or YwbH. Functions in acetic acid promotion of biofilm formation (Chen et al. 2015). Increases the activity of extracellular murein hydrolases probably by mediating their export via hole formation. May be inhibited by LrgB/YwbG (2.A.122.1.5).		CidA of Bacillus subtilis

1.E.14.1.17	Putative holin (or anti-holin) of 146 aas and 4 TMSs, YsbA or LrgA. YsbA is important for acetic acid induced biofilm formation (Chen et al. 2015) and for pyruvate utilization (van den Esker et al. 2016). van den Esker et al. 2016 proposed that YsbA is a pyruvate transporter. Thus, there is some question about the function of this protein and other members of this family.		YsbA of Bacillus subtilis

1.E.14.1.2	Holin, CidA (Bayles, 2003; Yang et al., 2005; Ranjit et al. 2011). Functions in biofilm formation in part by mediating release of cytoplasmic DNA during cell lysis to contribute to the biofilm matrix (Ranjit et al. 2011; Fischer et al. 2013). This protein contributes to cell lysis of dying cells (Patton et al. 2005). Calcium-chelating alizarin and other anthraquinones inhibit biofilm formation and modulate the expression of the cid/lrg genes (encoding the holin/antiholin system) (Lee et al. 2016). CidA and LrgA function as holins to support endolysin-induced lysis, and the lrgAB operon also facilitates pyruvate uptake during microaerobic and anaerobic growth (Laabei and Duggan 2022). The Staphylococcus aureus CidA and LrgA proteins are functional holins involved in the transport of by-products of carbohydrate metabolism (Endres et al. 2022).	Bacteria	*CidA of Staphylococcus aureus* (P60646)**

1.E.14.1.3	*Marine hydrolase exporter (MHE) (Desvaux et al.* 2005).**	Bacteria	*MHE of Fusobacterium mortiferum* (C3WD05)**

1.E.14.1.4	4 TMS LrgA putative holin/anti-holin. This protein has also been suggested to be a component of a 3-hydroxypropionate exporter, functioning together with YohK (TC# 2.A.1.122.1.1) (Nguyen-Vo et al. 2020).	Bacteria	*LrgA of E. coli* (F4V3T5)**

1.E.14.1.5	4 TMS murein hydrolase exporter, LrgA-like protein	Archaea	*LrgA-like protein of Thermococcus gammatolerans* (C5A1Q2)**

1.E.14.1.6	4 TMS Hypothetical protein (HP)	Archaea	*HP of Pyrococcus furiosus* (Q8TZY1)**

1.E.14.1.7	4 TMS LrgA-like protein	Bacteria	*LrgA-like protein of Chloroflexus aggregans* (B8GAY6)**

1.E.14.1.8	4 TMS holin protein	Archaea	*Holin of Pediococcus acidilactici* (D2EIF4)**

1.E.14.1.9	CidA holin-like protein	proteobacteria	*CidA of E. coli* (E7UC95)**

Examples:
TC#	Name	Organismal Type	Example
1.E.14.2.1	Putative archaeal holin of 145 aas and 4 TMSs	Euryarchaea	Putative holin of Methanocaldococcus (Methanococcus) vulcanius

1.E.14.2.2	Putative archaeal holin of 145 aas and 4 TMSs	Euryarchaea	Holin of Methanocaldococcus vulcanius

1.E.14.2.3	Putative holin of 194 aas and 5 TMSs	Euryarchaea	*Putative holin of Methanocaldococcus* sp.**